JPH07278191A - New peptide or protein and method for searching the same - Google Patents

Abstract

PURPOSE:To obtain the subject compound having such structure that the N- terminal and C-terminal of the amino acid sequence of a useful peptide or protein found in the natural world are turned reverse and the amino acid residues in the sequence are partly substituted, and serving as a inhibitor or promoter for the original compound, and useful for searching it. CONSTITUTION:This new compound contains e.g. amino acid sequences of respective formulas I-III, serving as e.g. a tumor necrosis factor inhibitor. This compound has such structure as to have the whole amino acid sequence or any two or more regions each made up of at least three amino acid residues constituting a mutually non-superposed portions of a partial amino acid sequence containing activity-manifesting region(s) with the N-terminal side and C-terminal side of the original amino acid sequence for at least one of said two or more regions turned reverse and also other amino acid residue (s) substituted for at least one amino acid residue. This compound has activity as an agonist, antagonist or inhibitor for the original peptide or protein.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to useful novel peptides or proteins and a method for efficiently searching them. Some of the peptides and the like of the present invention are tumor necrosis factor (TNF) -related peptides, and the present invention further provides
The present invention relates to a tumor necrosis factor inhibitor consisting of a peptide having an inhibitory activity on NF, or a physiologically acceptable salt thereof.

[0002]

2. Description of the Related Art The molecular weight isolated and purified from nature is about 1
In order to search for a novel peptide having a desired biological activity with reference to the amino acid sequences of not more than 10,000 biologically active peptides or derivatives thereof, first, amino acids are gradually deleted from the N-terminal side or C-terminal side of the natural peptide. It is known to synthesize short-chain peptides. By this method, short-chain peptides of several hormones have been synthesized so far. However, not all peptides can be made so short easily. For example, thyrotropin-releasing factor, luteinizing hormone-releasing factor (LHRH), oxytoci
n), vasopressin and angiotensin II are known to lose their activity when amino acids are deleted from their N-terminal and C-terminal sides, and the active region is composed of the entire amino acid sequence. Has been done.

Further, as a method for searching for a novel peptide based on a useful natural bioactive peptide, 1) an amino group at the N-terminus is an acetyl group or a carboxy group at the C-terminus is NH ₂ , NHMe, NHEt, etc. Protecting it as an amide, 2) substituting an amino acid residue at the N-terminal and an amino acid residue at the C-terminal with a non-natural amino acid residue, or introducing a non-natural amino acid in the middle of the peptide, 3) Peptide bond
Replacing (-CO-NH-) with -CH ₂ -NH-, -CH = CH-, -NH-CO-, or 4) searching for a new peptide by converting a linear peptide into a cyclic peptide Already known. as a result,
Many agonists, antagonists and inhibitors have been synthesized so far (AS Dutta; Advances in D
rug Research, 21, 145, 1991).

On the other hand, in the case of a protein having a molecular weight of about 10,000 or more, it is general to prepare a mutant having a desired activity by using a genetic engineering technique. However, in order to rapidly identify a binding region with a receptor or a ligand, it is a general method to make a mutant or synthesize a partial peptide and perform a binding experiment to identify it. Then, based on the primary structure or higher order structure of the identified binding region, an agonist, antagonist or inhibitor having a desired activity is searched for. So far, random synthesis has been developed as a method to search for them (JJ Devlin e
t al, 249, 404 (1990), SPA Fodor et al, Scienc
e, 251, 767 (1991), RA Houghtenet al, Peptide R
esearch, 5, 351, (1992), RW Frank et al, Peptid
e Research, 5, 315, (1992)). However, effective search guidelines have not yet been established.

With the development of molecular biology techniques, new peptides or proteins have been isolated from nature and their gene sequences (or amino acid sequences) have been identified.
Therefore, there is a demand for a method capable of simply and efficiently searching for a novel peptide having a novel agonist, antagonist or inhibitor activity on the basis of the amino acid sequence thereof.

[0006] Already, Shemyakin et al. Have used a topological approach (Angew. Chem.
Internat. Edit. 8, 492, (1969))
A dipeptide composed of at least one type of amino acid has been synthesized and at least one D-amino acid has been synthesized to evaluate its enzyme inhibitory activity. The dipeptide composed of that type of amino acid has a reverse sequence. Is not mentioned at all. Furthermore, Yamada et al. Reported a reverse sequence peptide of the peptide Arg-Gly-Asp-Ser which blocks the platelet adsorption of fibrinogen (KM Yamada et al, J. Cell. Bio
l., 99, 29 (1984)). That is, they also reported that the reverse peptide Ser-Asp-Gly-Arg blocks the adsorption of fibroblasts to fibronectin but not fibrinogen to platelets. In addition, such as offensive (C. Auffray
et al, WO8703601 (1987)) reported Arg-Phe-Asp-Ser and its reverse sequence peptide Ser-Asp-Phe-Arg as a peptide capable of blocking the binding between an antigen and T4 lymphocytes. Furthermore, from the amino acid sequence in the T4 molecule, Arg-Ala-Asp-
It has been described that the Ser sequence and its reverse sequence, Ser-Asp-Ala-Arg sequence, have binding-blocking ability. And a dimer peptide consisting of a reverse sequence peptide (for example, Arg-Ala-Asp-Ser-Ala-Ala-Ser-Asp-Ala-Arg). Recently, Fenton et al.
The reverse peptide of the pentapeptide (Thr-Arg-Ser-Ala-Trp), which has a bone resorption inhibitory activity called teostatin), was synthesized and the activity was evaluated, but it was inactive. (A.
J. Fenton et al, Endocrinology, 129, 3424 (1991)).
However, Maperi et al. Reported that the reverse peptide of magainin 2 has antibacterial activity (C. Mappelli et.
al, Eur. Pat. Appl. EP497366A2 (1992)).

However, the amino acid residues in a peptide in which only a part of the natural type amino acid sequence is reverse sequenced or in the natural type reverse sequence peptide have hitherto been described in the above amino acid classification method (glycine = alanine = proline). = Serine = threonine, phenylalanine = tyrosine = tryptophan, leucine = isoleucine = valine = methionine, lysine = arginine = histidine, glutamine = glutamic acid = asparagine = aspartic acid). No mention is made of the possibility of expressing.

On the other hand, JE Blalock et a
l, Proc. Natl. Acad. Sci. USA, 82, 1372, (1985))
Molecular Recognition Theo proposed by
based on ry), Shy et al. synthesized a complementary peptide (AS) to the naturally occurring ribonuclease S (RS) based on gene codons and reported that RS and AS actually bind. Furthermore, N of the amino acid sequence of the complementary peptide
It was also reported that a peptide in which the terminal side and the C-terminal side were reversed (that is, a reverse complementary peptide) (IAS) also binds to RS (Y.
Shai et al, Biochemistry, 26, 669, (1987)). Regarding such an interaction between a sense peptide and a complementary peptide or a sense peptide and an anti-complementary peptide, Alu-Obage et al. Reported a similar example (FA Al-Obeid
i et al, Peptide, Chem. Struct. Biol. Proc. Am. Pe
p. Symp. 11th, 530, (1989)). However, whether they are effective in synthesizing and evaluating reverse sequence peptide derivatives of naturally occurring sense peptides according to the above-mentioned amino acid classification as a means for searching for new agonists, antagonists or inhibitors. Does not mention at all.

[0009] By the way, TNF is a kind of cytokine having many actions on cells and binds to a specific cell surface receptor to start its action. TNF kills tumor cells and virus-infected cells and is expected to be effective in treating cancer.

However, in some diseases, TNF exerts harmful effects such as septic shock, anorexia and weight loss due to its overproduction. From this, TNF, which is also called cachectin, is reported to be a causative agent of cachexia [B. Beutler et al., Nature, 316, 552 (198).
5) and KJ Tracey et al., Nature, 330, 662 (198).
7)]. In addition, recently, TNF is a causative virus of acquired immunodeficiency syndrome (AIDS), human immunodeficien.
Along with the action of amplifying cy virus (HIV) replication, HIV
Target cells, helper / inducer T-cells (CD4
When the expressing cells are infected with HIV, TNF selectively
The action of killing D4 cells and promoting immunodeficiency was observed [Pharmaceutical Journal, 113, 1-18 (1993)]. From this, the endogenously formed TNF or the exogenously administered T
It is an important task to appropriately control NF and find a substance that antagonizes or inhibits these side effects.

As a substance exhibiting a TNF inhibitory action, TBP-I from human urine [I. Olsson et al., Eur. J. Haematol., 42, 27]
0 (1989) and H. Engelmann, J. Biol. Chem., 26.
4, 11974 (1989)] and TBP-II [H. Engelmann et al., J.
Biol. Chem., 265, 1531 (1991)] about 30 kD
A TNF-binding protein having a molecular weight of
It has been identified that it corresponds to the extracellular domain of the F receptor protein and is reported to exhibit an inhibitory effect on the TNF effect. These TNF receptor partial proteins and other protein preparations such as anti-TNF monoclonal antibodies are expected as therapeutic agents for endotoxin shock associated with sepsis or therapeutic agents for AIDS.

[0012] 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. Obtaining a refined product requires special equipment and skilled techniques, and it takes a long period of time and a lot of labor. Therefore, with conventional proteins, the cost is high, the TNF antagonist or inhibitor produced is expensive, and mass production is difficult. Currently, there are no TNF antagonists or inhibitors on the market.

DISCLOSURE OF THE INVENTION The present inventor has keenly sought a method for conveniently and efficiently searching for a novel peptide having an action or an antagonistic action similar to that of a useful biologically active peptide or a peptide in the functional region of a protein. As a result of the investigation, surprisingly, a peptide or protein in which partial or complete reverse sequences of two or more partial sequences of the original amino acid sequence are linked, or a peptide or protein in which a partial amino acid of the reverse sequence peptide is substituted, The inventors have found that they can exhibit useful biological activity as agonists, antagonists or inhibitors of peptides or proteins having the original sequence, and completed the present invention. Furthermore, the inventors of the present invention searched for a synthetic peptide having inhibitory activity against TNF and capable of being mass-produced at low cost by using the above-mentioned method, and as a result, the short-chain synthetic peptide of the present invention was identified. I found it.

[0013]

Means for Solving the Problems The present invention is directed to the overlapping of the entire amino acid sequence of a peptide or protein consisting of 6 or more amino acids with a useful biological activity or a partial amino acid sequence containing a region expressing the activity. It is composed of any two or more regions consisting of three or more amino acid residues constituting a part thereof, and any one or more or all of these regions have the N-terminal side and the C-terminal side of the original amino acid sequence. A peptide or protein that has an inverted amino acid sequence and exhibits agonistic, antagonistic, or inhibitory activity of a peptide or protein having the above-mentioned useful biological activity (provided that the reverse sequence of the original amino acid sequence is completely Peptides or proteins with matching amino acid sequences are excluded, and the N-terminal amino group of the peptide is Butyl group, may be modified by the t- butoxycarbonyl group or benzyloxycarbonyl groups, C
The terminal carboxyl group may be an amide group. Further, when two cysteines are present in the peptide, they may combine to form an intramolecular disulfide bond). However, when a cysteine residue is present in the amino acid sequence, the cysteine residue is glycine, alanine, proline, serine, threonine, phenylalanine, tyrosine, tryptophan, leucine, isoleucine, valine, methionine, lysine, arginine, It may be substituted with an amino acid residue selected from the group consisting of histidine, glutamine, glutamic acid, asparagine, aspartic acid, S-acetamidomethyl-cysteine, and α-aminoisobutyric acid.

Specific examples of the above peptides or proteins include SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 1.
0, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, or the peptide or protein represented by SEQ ID NO: 14 (however, two Cys in the peptide of SEQ ID NO: 13 bind to each other to form an intramolecular disulfide bond). Can be mentioned.

Furthermore, the present invention provides a complete amino acid sequence of a peptide or protein consisting of 6 or more amino acids having a useful biological activity or a partial amino acid sequence containing a region expressing the activity, which does not overlap with each other. Assuming a region obtained by dividing it into any two or more regions consisting of three or more amino acid residues constituting it; and then comprising two or more regions among those regions, any one or more or all of them At least 1 in the amino acid sequence in which the N-terminal side and the C-terminal side of each original amino acid sequence are reversed, or in the reversed amino acid sequence.
Method for searching useful peptide or protein characterized by synthesizing peptide or protein derivative having amino acid sequence in which amino acid residue of residue is substituted with same number of other amino acid residue, and further evaluating biological activity thereof I will provide a. However, the above-mentioned other amino acid residues are group I (glycine, alanine, proline, serine, threonine), group II (phenylalanine, tyrosine, tryptophan), group I.
II (leucine, isoleucine, valine, methionine),
If it belongs to either Group IV (lysine, arginine, histidine) or Group V (glutamine, glutamic acid, asparagine, aspartic acid), it is an amino acid residue selected from the other amino acids in the group to which it belongs, and When a cysteine residue is present in the amino acid sequence, the cysteine residue is glycine, alanine, proline, serine, threonine, phenylalanine, tyrosine, tryptophan, leucine, isoleucine, valine, methionine, lysine, arginine, histidine, It may be substituted with an amino acid residue selected from the group consisting of glutamine, glutamic acid, asparagine, aspartic acid, S-acetamidomethyl-cysteine, and α-aminoisobutyric acid.

The present invention also provides the entire amino acid sequence of a peptide or protein having a useful biological activity isolated from the natural world, or the N-terminal side of an amino acid sequence consisting of 3 or more amino acid residues and exhibiting the activity. A peptide or protein having an amino acid sequence in which the C-terminal side is reversed and at least one amino acid residue is replaced with the same number of other amino acid residues, and the original peptide or peptide having useful biological activity A peptide or protein having protein agonist, antagonist, or inhibitor activity (wherein the N-terminal amino group of the peptide is an acetyl group,
It may be modified with a t-butoxycarbonyl group or a benzyloxycarbonyl group, and the C-terminal carboxyl group may be an amide group. Further, when two cysteines are present in the peptide, they may combine to form an intramolecular disulfide bond). However,
The other amino acid residue is a group I (glycine, alanine, proline, serine,
Threonine), group II (phenylalanine, tyrosine, tryptophan), group III (leucine, isoleucine, valine, methionine), group IV (lysine, arginine, histidine), or group V (glutamine, glutamic acid, asparagine, aspartic acid). When it belongs to, it is an amino acid residue selected from other amino acids in the group to which it belongs,
When the amino acid residue to be replaced is a cysteine residue, any of the amino acids of Group I to Group V above,
Alternatively, it is an amino acid residue selected from S-acetamidomethyl-cysteine and α-aminoisobutyric acid. Specific examples of these peptides or proteins include SEQ ID NO: 10,
Sequence number 11, sequence number 12, sequence number 13, sequence number 14, sequence number 15, sequence number 16, or sequence number 17
The peptide or protein shown by (however, SEQ ID NO: 1
2 cys in the peptide of 3 may combine with each other to form an intramolecular disulfide bond).
Furthermore, the present invention provides the N-terminal side and C-terminal side of the amino acid sequence of a peptide or protein having a useful biological activity isolated from nature or an amino acid sequence consisting of three or more amino acid residues which express the activity. And a peptide or protein having an amino acid sequence in which at least one amino acid residue is replaced with the same number of other amino acid residues, and the biological activity thereof is evaluated. A method for searching a peptide or protein is provided. However, the above-mentioned other amino acid residue means that the amino acid residue to be replaced is group I (glycine, alanine, proline, serine, threonine), group II.
(Phenylalanine, tyrosine, tryptophan), group III (leucine, isoleucine, valine, methionine), group IV (lysine, arginine, histidine) or group V (glutamine, glutamic acid, asparagine, aspartic acid) If the amino acid residue selected from the other amino acids in the group to which it belongs and the amino acid residue to be replaced is a cysteine residue, the group from the above group I to
Any of the amino acids of V, or S-acetamidomethyl-
It is an amino acid residue selected from cysteine and α-aminoisobutyric acid. The present invention further provides a tumor necrosis factor inhibitor, a nerve growth factor production promoter, a cell adhesion inhibitor, or an antibacterial agent, which comprises the above peptide or a physiologically acceptable salt.

55 as a peptide having a useful biological activity in the present invention and consisting of amino acids having a certain number of residues or more.
TNF binding region of kdTNF (tumor necrosis factor) receptor, 55kdTNF receptor-derived nerve growth factor (nerve g
A rowth factor producing region, an antibacterial peptide (magainin 2), a cell adhesion peptide (RGD), and the like will be specifically described, and in addition, adrenocorticotropic hormone (adrenocortic hormone)
otropic hormone), adrenocorticotropic hormone releasing factor (c
orticotropin-releasing factor), angiotensin I
I (angiotensin II), natriuretic hormone (atria
l natriuretic factor), bombestin (bombesti
n), bradykinin, calcitonin (cal)
citonins), calcitonin-related peptide (calcitonin g
ene-related peptide), cholecystokinin (cholecyst)
okinin), cecropin (cecropin A), dermorphin (dermorphin), dynorphin (dynorphin A), enkephalin (enkephalin), endothelin (endothel)
ins), gastrin (gastrin), gastrin-releasing peptide (gastrin-releasing peptide), glucagon (gluca)
gon), growth hormone-releasing peptide (growth hormone-rel
easing peptide), insulin A-chai
n), insulin B-chain, leutenizing hormone-releasing ho
rmone), magainin I, melanocyto-stimulating hormone r
eleasing-inhibiting factor, melittin
n), neurokinin A and B (neurokinin A and B), neurotensin (neurotensin), neutrophil defensin (neutrophil defensin), oxytocin (o
xytocin), secretin, somatostatin, substance P, tachyplesin, thyrotropin-releasing factor, vasopressin, and the like. Acquired immunodeficiency (HIV), poliovirus (Poliovirus) and B
Binding regions for the envelope proteins of viruses such as hepatitis B virus, and interferons and interleukins
Examples include binding regions to receptors such as various cytokines such as. Furthermore, the present invention can be applied to ligand binding regions of various receptors and enzyme inhibitor peptides. In addition, the present invention can be basically applied to the construction of reverse sequences of various high molecular weight proteins by using gene synthesis and genetic engineering techniques. Further, by utilizing the concept of the present invention, a protein having a known biological activity having a homology of about 20% or more with a reverse amino acid sequence or a positive amino acid sequence of a protein of unknown biological activity under the homology condition using the above amino acid classification method is used. It can be inferred that a protein with unknown biological activity can be evaluated by selecting a normal amino acid sequence or a reverse amino acid sequence from an existing database.

[0018] In the natural world, melanocyto-stimulating hormone releasing-inh
ibiting factor), thyrotropin-releasing factor (thyr
otropin-releasing factor), cell adhesion peptide (RG
Useful peptides consisting of three amino acid residues such as D) are known. Therefore, from the gist of the present invention, 20
Even if a peptide consisting of at least three or more amino acid residues is synthesized by random synthesis using various kinds of amino acids,
The amino acid sequence of the synthesized peptide is the amino acid taxonomy according to the claims (glycine = alanine = proline = serine =
Threonine, phenylalanine = tyrosine = tryptophan, leucine = isoleucine = valine = methionine,
Homology conditions using lysine = arginine = histidine, glutamine = glutamic acid = asparagine = aspartic acid) The reverse sequence of the amino acid sequence of naturally occurring active peptide and other amino acid residues except cysteine have the same homology. If so, it is possible to fully speculate that the synthesized peptide has agonistic or antagonistic activity of the naturally occurring active peptide. This confirmed that possibility in the example relating to SEQ ID NO: 17. Further, regarding the possibility in the case of a useful peptide consisting of 4 or more amino acid residues, as can be seen from the examples of Magainin 2 for example, even if it is synthesized by a random synthesis method using 20 kinds of amino acids, When the amino acid sequence of the synthesized peptide has homology except for cysteine with a part of the reverse sequence of the amino acid sequence of the naturally occurring active peptide under homology conditions using the above-mentioned amino acid classification method, the synthesis It supports the possibility that the obtained peptide may have agonistic or antagonistic activity of the naturally occurring active peptide. Therefore, the derivative of the reverse sequence peptide or reverse sequence protein obtained by the random synthesis method is also within the scope of the present invention.

For the synthesis of the peptide or protein of the present invention,
A liquid phase method, a solid phase method or a genetic engineering method may be used as necessary. It is obvious that when a desired peptide or protein is searched for by genetic engineering using a prokaryotic cell or a eukaryotic cell, it is based on the principle necessary for expressing a heterologous gene. In the case of a synthetic peptide by the liquid phase method or the solid phase method, the N-terminal amino group may be modified with an acetyl group, butoxycarbonyl group, benzyloxycarbonyl group, etc., and the C-terminal carboxyl group is an amide group. May be. However, cysteine in the amino acid sequence may be synthesized as it is, but may be substituted with an amino acid such as glycine or serine for the sake of simplicity of synthesis and purification, and S-acetamidomethyl-cysteine (if necessary) S-acetam
idomethyl-cysteine), α-aminoisobutyric acid (α-amino
It may be replaced with isobutylic acid) or the like. Furthermore, those synthetic peptides or proteins may be physiologically acceptable salts.

As physiologically acceptable salts, 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, With 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 Examples thereof include salt.

As specific examples of the peptide or protein of the present invention, SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 4 or SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13 as shown in Examples. , SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16 or the peptide represented by SEQ ID NO: 17.

The method for evaluating the biological activity is according to a known method for evaluating the biological activity of the peptide or protein, and depending on the purpose, in vitro or in vivo.
It can be measured in o). For example, TNF binding region of 55kd TNF receptor, nerve growth factor production region derived from 55kd TNF receptor, magainin 2
Alternatively, the method for evaluating the activity of the cell adhesion peptide or the like is described in each Example.

Thus, whether it is a useful peptide or protein
To find a peptide having the desired activity, and then
Biological activity of peptides (agonists, antagonists,
Inhibitor activity) increase / decrease, solubility, stability in blood, etc.
Those skilled in the art will be able to synthesize derivatives for the purpose and evaluate their activity.
It is easy to guess. Specifically, for the peptide
N-terminal acetylation, C-terminal amidation,
Bond (-CO-NH-) to -CH ₂-NH-, -CH = CH-, -NH-CO-
W. V. Williams et a
l, J. Biol. Chem., 266, 5182 (1991))
Is it a linear type by introducing two cysteine residues at appropriate positions?
To make a circular shape, or convert to the opposite direction,
Or exchange of L-amino acid residue with D-amino acid residue, etc.
The modification method by is a known method.

The peptide of the present invention can be easily obtained by applying a general peptide synthesis method. Peptide synthesis methods include activated ester method, mixed acid anhydride method, C-terminal activation method such as azide method, coupling method such as carbodiimide, N-carboxyanhydride (NCA) method, redox method,
Enzymatic method or solid-phase method developed by Merrifield [RB
Merrifield, Angew. Chem. Int. Ed. Engl., 24, 799
(1985)] etc. In addition, 2
The ligation of individual cysteine residues is performed by an oxidation reaction using air, iodine, dimethylsulfoxide or the like.

The tumor necrosis factor inhibitor, nerve growth factor production promoter, cell adhesion inhibitor or antibacterial agent in the present invention can be administered orally or parenterally for treatment.

The oral administration agent may be a solid preparation such as powder, granules, capsules and tablets, or a liquid preparation such as syrup and elixir. In addition, parenteral administration agents such as injections, rectal administration agents, external preparations for skin and inhalants can be used. These formulations are manufactured in a conventional manner by adding to the active ingredient a pharmaceutically acceptable manufacturing auxiliary agent. Further, it is also possible to prepare a sustained-release preparation by a known technique.

To prepare solid preparations for oral administration, active ingredients and excipients such as lactose, starch, crystalline cellulose,
Lactose calcium, magnesium aluminometasilicate,
Mix with silicic acid anhydride etc. to make a powder, or if necessary, add a binder such as sucrose, hydroxypropylcellulose, polyvinylpyrrolidone, etc., a disintegrating agent such as carboxymethylcellulose, carboxymethylcellulose calcium, etc. Granulate into granules. To manufacture tablets, these powders and granules may be tableted as they are or by adding a lubricant such as magnesium stearate and talc. These granules or tablets are coated with an enteric base such as hydroxypropylmethylcellulose phthalate, methacrylic acid, and methyl methacrylate copolymer to form an enteric preparation, or ethylcellulose, carnauba wax, hardened oil, etc. to form a sustained-release preparation. You can also do it. In order to produce capsules, hard capsules such as powder or granules are filled, or the active ingredient is glycerin, polyethylene glycol, sesame oil,
It can be dissolved in olive oil or the like and then coated with a gelatin film to give a soft capsule.

To prepare a liquid preparation for oral administration, an active ingredient and a sweetener such as sucrose, sorbitol or glycerin are dissolved in water to prepare a transparent syrup, and essential oil, ethanol or the like is added to form an elixir. Or gum arabic,
An emulsion or suspension may be prepared by adding tragacanth, polysorbate 80, sodium carboxymethyl cellulose and the like. If desired, flavoring agents, coloring agents, preservatives and the like may be added to these liquid preparations.

In order to produce an injectable preparation, an active ingredient may be added as necessary to a pH adjusting agent such as hydrochloric acid, sodium hydroxide, emulsion, sodium lactate, sodium monohydrogen phosphate and sodium dihydrogen phosphate, sodium chloride, glucose and the like. It is also dissolved in distilled water for injection with an isotonicity agent, aseptically filtered and filled in an ampoule, or mannitol, dextrin, cyclodextrin, gelatin, etc. are added and freeze-dried under vacuum to prepare a solution-soluble injection. Alternatively, lecithin, polysorbate 80, polyoxyethylene hydrogenated castor oil and the like may be added to the active ingredient and emulsified in water to give an emulsion for injection.

In order to produce a rectal preparation, the active ingredient and a suppository base such as cocoa butter, fatty acid tri-, di- and monoglycerides, polyethylene glycol and the like are moistened and melted and poured into a mold and cooled, or The active ingredient may be dissolved in polyethylene glycol, soybean oil or the like and then coated with a gelatin film.

To prepare an external preparation for the skin, the active ingredient is added to white petrolatum, beeswax, liquid paraffin, polyethylene glycol, etc., and if necessary, moistened and kneaded to form an ointment, or rosin, an alkyl acrylate ester After kneading with an adhesive such as coalescence, it is spread on a non-woven fabric such as polyethylene to form a tape.

To produce an inhalant, the active ingredient is dissolved or dispersed in a propellant such as CFC gas and filled in a pressure resistant container to form an aerosol.

The dose of the peptide of the present invention depends on the age of the patient,
Usually about 1 mg to 5 per day, depending on body weight and condition
It is 00 mg, and it is desirable to administer it in 1 to several divided doses.

[0034]

EXAMPLES The present invention will be described in more detail with reference to Examples and Test Examples. However, the present invention is not limited to these.

Example 1 Peptide Synthesis Peptide synthesis was carried out using an automated peptide synthesizer (Applied Biosyst
430 A or 431 A manufactured by ems) was used for solid phase synthesis. The amino acids used in the synthesis were Boc-Ala and Boc-Arg (Mt
s), Boc-Asn, Boc-Asp (OBzl), Boc-Cys (4-CH3OBzl), Bo
c-Gln, Boc-Glu (OBzl), Boc-Gly, Boc-His (Dnp), Boc-I
le, Boc-Leu, Boc-Lys (Cl-Z), Boc-Met (O), Boc-Phe, B
oc-Pro, Boc-Ser (Bzl), Boc-Thr (Bzl), Boc-Trp (CHO),
Boc-Tyr (Br-Z) and Boc-Val. PAM resin was used for C-terminal carboxylic acid type peptide synthesis, and P-methyl BHA resin was used for C-terminal amide type peptide synthesis. Removal of the protecting group and cleavage from the resin followed the manual of Applied Biosystems. The salts contained in the crude peptide after excision were removed using an open column [Bio-Gel P-2], 1
It was carried out using a 0% acetic acid aqueous solution. Then, in the case of a linear peptide, the crude peptide was subjected to HPLC [HPLC [ODS-80T
Using M column (Tosoh, 21.5 mm ID x 30 cm), acetonitrile linear gradient / 0.1% trifluoroacetic acid],
The desired peptide was lyophilized. In the case of a cyclic peptide, after desalting, a cyclization reaction such as air oxidation, I ₂ oxidation or DMSO oxidation was performed, and then the desired peptide was fractionated using an ODS-80TM column. The purified peptide is an analytical column [ODS-80TM column (Tosoh, 4.6 mm ID x 15c
m), acetonitrile linear gradient / 0.1% trifluoroacetic acid)
After making sure it is single using Applie
Amino acid sequence analysis was performed using a 477A gas phase protein sequencer manufactured by Biosystems. In addition, the amino acid composition analysis of the peptide was carried out using an A-8700 type amino acid analyzer (Ichika Kogyo). The synthesized peptides are shown in Table 1.

[0036]

[Table 1] Table 1 Synthetic peptides ──────────────────── SEQ ID NO: 1 LNGTVHLSAGFFLRENE-NH ₂ SEQ ID NO: 2 LNGTVHLSENERLFFGA-NH ₂ SEQ ID NO: 3 SLHVTGNLAGFFLRENE-NH ₂ SEQ ID NO: 4 SLHVTGNLENERLFFGA-NH ₂ SEQ ID NO: 5 SAQEKQNTLRENE-NH ₂ SEQ ID NO: 6 ENERLTNQKEQAS-NH ₂ SEQ ID NO: 7 SAQEKQNTENERL-NH ₂ SEQ ID NO: 8 TNQKEQASLRENE-NH ₂ SEQ ID NO: 9 TNQKEQASENERL-NH ₂ ─────────── ─────────────

The peptides of SEQ ID NOs: 1 to 9 are 55 kdTN
TNF binding region of F receptor [BL.Lie et al, Bioche
m, Biophhys.Res.Commun., 188, 503 (1992)] is a derivative synthesized in accordance with the gist of the present invention.

Example 2 Synthesis of Peptide Represented by SEQ ID NO: 10 The solid phase method was used for peptide synthesis, and an automatic peptide synthesizer was used.
(Applied Biosystems model 430 A or 431 A)
Was carried out according to the company's program. If the reagent used in the synthesizer is 430 A, 1. Dipropylamineamine 2. Trifluoroacetic acid 3. Nutrizer
(Ethanolamine) 4. Methanol 5. Hydroxybenzotriazole 6. Dicyclohexylcarbodiimide 7. Dichloromethane 8. Dimethylformamide. In the case of 431 A, instead of dimethylformamide,
N-methylpyrrolidone was used. The amino acids used in the synthesis are Boc-Ala, Boc-Arg (Mts), Boc-Asn, Boc-
Asp (OBzl), Boc-Cys (4-CH3OBzl), Boc-Glu (OBzl), Boc
-Gln, Boc-Gly, Boc-His (Dnp), Boc-Leu, Boc-Lys (Cl-
Z), Boc-Phe, Boc-Ser (Bzl), Boc-Thr (Bzl), Boc-Trp (C
HO), Boc-Tyr (Br-Z) and Boc-Val. PAM resin was used for C-terminal carboxyl group type peptide synthesis, and P-methyl BHA resin was used for C-terminal amide group type peptide synthesis. Applied for removal of protecting groups and cutting out from resin
The Biosystems manual was followed.

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 2.6 g of peptide resin was obtained. The above peptide resin was suspended in dimethylformamide (50 ml), thiophenol (2.6 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 (2.1 ml) and dimethyl sulfide (7.8 ml) were added, and trifluoroacetic acid (13.0 ml) and then trifluoromethanesulfonic acid (2.6 ml) were added to the stirred mixture while cooling in an ice bath. It was 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 in the resulting mixture:
1,2-ethanedithiol (2: 1) (3.9 ml) was added, and the mixture was stirred for 10 minutes. Then trifluoroacetic acid (26 ml) was added while cooling in an ice bath. Trifluoromethanesulfonic acid
(2.6 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 treated with trifluoroacetic acid.
It was dissolved in (15 ml), cooled, and added dropwise to the stirring ether. The precipitate was filtered and washed 3 times with ether.
It was dissolved in a 10% aqueous acetic acid solution and filtered. This 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 1.68 g of a white powder. HPLC [ODS-
It was fractionated by 80TM column (Tosoh, 21.5 mmID x 30 cm), acetonitrile linear gradient / 0.1% trifluoroacetic acid] and freeze-dried to obtain a white powder (500 mg). Analytical column [ODS-80
HPLC was carried out using a TM column (Tosoh, 4.6 mmID x 15 cm) acetonitrile linear gradient / 0.1% trifluoroacetic acid], and it was confirmed that the purified product was single. Applied part
Biosystems 477A Protein Sequencer, 120A
Amino acid sequence analysis was performed using a PTH analyzer. After hydrolysis with 6N hydrochloric acid at 110 ° C for 20 hours,
The analysis was carried out using an I-8700 amino acid analyzer manufactured by Ichikaika. Based on the analysis results, the peptide sequence is Gln Phe Leu Asn.
Glu Ser Trp Tyr His Arg Tyr Gln Asn Lys
Arg Ala Gly Ala Val Thr Asp Arg Asp Val
It was confirmed to have Thr Gly. This peptide is a 55kd TNF receptor [H. Loetscher et al, Cel
1, 61, 351 (1990)] in the 117-142 region (amino acid sequence: CTVDRDTVCGCRKNQYRHYWSENLFQ).

Examples 3 to 4 The same or similar reaction procedure as in Example 1 was carried out to obtain the peptides shown in Table 2.

[0042]

[Table 2] Table 2 Synthetic peptides ──────────────────────── SEQ ID NO: 11 ENERLFFGAHATAVTNQKEQG SEQ ID NO: 12 HATAVTNQKEQASLHVTGNLALS ────────── ─────────────── SEQ ID NOS: 11 and 12 are TN of 55kd TNF receptor
F binding region (amino acid sequence: SLCLNGTVHLSCQEKQNTVCTCH
And QEKQNTVCTCHAGFFLRENE) (BL. Lie et al, Biochem.
Biophys. Res. Commun., 188, 503 (1992)].

Example 5 Synthesis of peptide represented by SEQ ID NO: 13 Boc-Cys (4-CH ₃ OBzl) -PAM-resin was used in the same manner as in Example 2 to obtain 1.38 g of a peptide resin. . Suspended in dimethylformamide (33 ml), added thiophenol (2.8 ml), and stirred for 3.5 hours. After filtration, the peptide resin was washed with dichloromethane. Hereinafter, deprotection and cutting out from the resin were performed by a general method. Thioanisole on peptide resin
(1.4 ml) and 1,2-ethanedithiol (0.7 ml) were added, and trifluoroacetic acid (14 ml) and trifluoromethanesulfonic acid (1.4 ml) were added with stirring under ice cooling. After 30 minutes, diethyl ether (100 ml) was added. The precipitate was filtered and washed with diethyl ether. Trifluoroacetic acid on the precipitate
(5 ml) was added to dissolve the peptide, and this trifluoroacetic acid layer was added dropwise to 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. Lyophilization gave a powder (250 mg). This powder was prepared by a conventional method [A. Otaka et al, Tetrahedron Lett., 32, 1223 (19
91)] and dissolved in a mixed solution of dimethylsulfoxide (2.5 ml) and trifluoroacetic acid (250 ml), and the mixture was stirred for 12 hours. The solvent was distilled off under reduced pressure with a water stream, and the residue was purified by HPLC to obtain 57 mg of the peptide of Example 5 (SEQ ID NO: 13). A part was subjected to amino acid sequence analysis using a 477A protein sequencer and 120A PTH analyzer manufactured by Applied Biosystems. SEQ ID NO: 13 is the TNF binding region of 55 kdTNF receptor (amino acid sequence: CQEKQNTVCTCH)
[BL. Lie et al, Biochem. Biophys. Res. Commun., 1
88, 503 (1992)].

Example 6 The same reaction procedure as in Example 1 was carried out to obtain the peptides shown in Table 3.

[0045]

Table 3 Table 3 Synthetic peptides ──────────────────── SEQ ID NO: 14 AKGAKKASHLWKGIG-NH ₂ SEQ ID NO: 15 SDPR-NH ₂ SEQ ID NO: 16 SNGR-NH ₂ SEQ ID NO: 17 NGR-NH ₂ ────────────────────

SEQ ID NO: 14 is magainin 2-amide (amino acid sequence: GIGKFLHSAKKFGKAFVGEIMSN-NH ₂ ) [MA Z
asloff, PB88-106778 (1987)]. SEQ ID NO: 15, SEQ ID NO: 16, and SEQ ID NO: 1
7 is a cell adhesion peptide (amino acid sequence: RGDS) [KM Y
amada et al, J. Cell. Biol., 99, 29 (1984)].

Test Example 1 TNF Cell Damage Inhibitory Activity Measurement Test Mouse LM cells were cultured in Dulbecco's Modified Eagle Medium (DMEM) containing 5% bovine serum and 0.2% glucose. A solution prepared by pre-culturing TNF alone or a peptide and TNF at 37 ° C. for 18 hours was cultured on a 96-well microplate at 0.8 to 1 × 10 ⁴ cells / well for 1 day.
-Added to M cells. After 24 hours, each well was stained with crystal purple. The TNF cytotoxicity-suppressing activity of the peptide was determined by extracting the dye absorbed in cells and measuring the absorbance at 550 nm using a microplate reader.
It was calculated by comparing with the absorbance of wells in which F alone was added and the medium was cultured only with the culture solution. The suppression value of the peptide was calculated from the value of the absorbance when the mixture of the peptide and TNF was added, with the suppression of the absorbance value of the well to which TNF alone was added as 0% and the suppression of the absorbance value of the well cultured only with the culture solution as 100%. .

[0048]

Table 4 Table 4 Effect of synthetic peptides on the cytotoxic activity of TNF on LM cells ──────────────────────────── ─── TNF cytotoxicity inhibitory activity (%) ────────────────────────────── TNF (3.5ng / ml) 0 TNF (3.5ng / ml) + SEQ ID NO: 1 (1mM) ND ^* TNF (3.5ng / ml) + SEQ ID NO: 2 (1mM) 65 TNF (3.5ng / ml) + SEQ ID NO: 3 (1mM) 64 TNF (3.5ng / ml) + SEQ ID NO: 4 (1mM) 39 TNF (3.5ng / ml) + SEQ ID NO: 5 (1mM) 2 TNF (3.5ng / ml) + SEQ ID NO: 6 (1mM) 3 TNF (3.5ng / ml) + SEQ ID NO: 7 (1mM) 4 TNF (3.5ng / ml) + SEQ ID NO: 8 (1mM) 4 TNF (3.5ng / ml) + SEQ ID NO: 9 (1mM) 0 ─────────────── ─────────────── ND ^* ； Not measured

The results in Table 4 show that the cytotoxic activity of TNF is suppressed in the presence of the synthetic peptides of SEQ ID NOs: 2-4. On the other hand, it is shown that the cytotoxic activity of TNF is not suppressed in the coexistence of the synthetic peptides of SEQ ID NOs: 5 to 9. Therefore, using these two methods,
It is shown that the binding site in the bioactive expression region can be further specified.

Test Example 2 TNF Cell Damage Inhibitory Activity Measurement Test The procedure is the same as in Test Example 1.

[0051]

[Table 5] Table 5. Effect of synthetic peptides on the cytotoxic activity of TNF on LM cells ───────────────────────────── ─────── TNF cell damage suppression (%) ─────────────────────────────────── TNF (3.5ng / ml) 0 TNF (3.5ng / ml) + SEQ ID NO: 10 (1mM) 56 TNF (3.5ng / ml) + SEQ ID NO: 11 (1mM) 46 TNF (3.5ng / ml) + SEQ ID NO: 12 (1mM) ) 53 ───────────────────────────────────

The results in Table 5 show that the cytotoxic activity of TNF was suppressed in the presence of the synthetic peptides of SEQ ID NOs: 10-12.

Test Example 3 NGF (nerve growth factor) production promoting activity measurement L of 2 × 10 ⁴ cells / ml (199 medium containing 0.5% peptone)
-The M cell suspension was cultivated in a 96-well multiplate at 0.2 ml per well for 3 days, and the peptide (SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4) dissolved in 199 medium containing 0.5% BSA was used. , SEQ ID NO: 10 or SEQ ID NO: 19
The solution was exchanged with the medium, and the cells were further cultured for 24 hours. After the culture was completed, NGF contained in the culture was measured by the EIA method using an anti-mouse NGF monoclonal antibody.
As a result, it shows in FIGS. The peptides of SEQ ID NO: 10, SEQ ID NO: 19, SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 4 promoted NGF production in a concentration-dependent manner.

Test Example 4 Antibacterial Activity Measurement A single colony of E. coli AD31 on an LB plate was suspended in 5 ml of Mueller Hinton medium (MH) and cultured at 30 ° C. overnight. 1 of 96-well microplate (SUMITOMO)
100 μl of a bacterial suspension of overnight culture diluted to 1 × 10 ⁶ cells / ml with MH per well, 80 μl of MH medium, and 20 μl of a peptide sample dissolved in secondary pure water were mixed. The bacteria were cultured by leaving the 96-well microplate in a thermostat at 30 ° C. for 24 hours. Absorbance of culture suspension (6
30nm) microplate reader (CORONA ELECTRIC MT
P-32).

[0055]

[Table 6]

Test Example 5 Measurement of Cell Adhesion Inhibitory Activity A375 cells (human malignant melanoma-derived cells) contained 10% FB
Dulbecco's Modified containing S and 0.45% glucose
Cultured with Eagle's Medium. The cells in the logarithmic growth phase were detached from the culture flask by trypsin, and the cells were washed with 0.1% BS.
It was washed twice with PBS containing A. Then 0.1%
A cell suspension was prepared from a peptide solution dissolved in Dulbecco's Modified Eagle's Medium containing BSA, and cells and peptides were sensitized at 37 ° C. for 10 minutes. Then, in advance, 1 μg of fibronectin / well / 0.1 ml of 3
96-well ELISA plate (N
unc, Immunoplate) was added with 5 × 10 ⁴ cell suspensions per well and incubated at 37 ° C. for 30 minutes. After the completion of the culture, each well was washed three times with PBS and stained with 0.5% crystal violet, and then the dye was extracted and the absorbance (550 nm) was measured.

[0057]

[Table 7]

[0058]

[Sequence list]

SEQ ID NO: 1 Sequence length: 17 Sequence type: Amino acid Topology: Linear Sequence type: Peptide sequence Leu Asn Gly Thr Val His Leu Ser Ala Gly Phe Phe Leu Arg Glu Asn 1 5 10 15 Glu

SEQ ID NO: 2 Sequence length: 17 Sequence type: Amino acid Topology: Linear Sequence type: Peptide sequence Leu Asn Gly Thr Val His Leu Ser Glu Asn Glu Arg Leu Phe Phe Gly 1 5 10 15 Ala

SEQ ID NO: 3 Sequence length: 17 Sequence type: Amino acid Topology: Linear Sequence type: Peptide sequence Ser Leu His Val Thr Gly Asn Leu Ala Gly Phe Phe Leu Arg Glu Asn 1 5 10 15 Glu

SEQ ID NO: 4 Sequence length: 17 Sequence type: Amino acid Topology: Linear Sequence type: Peptide sequence Ser Leu His Val Thr Gly Asn Leu Glu Asn Glu Arg Leu Phe Phe Gly 1 5 10 15 Ala

SEQ ID NO: 5 Sequence length: 13 Sequence type: Amino acid Topology: Linear Sequence type: Peptide sequence Ser Ala Gln Glu Lys Gln Asn Thr Leu Arg Glu Asn Glu 1 5 10

SEQ ID NO: 6 Sequence length: 13 Sequence type: Amino acid Topology: Linear Sequence type: Peptide sequence Glu Asn Glu Arg Leu Thr Asn Gln Lys Glu Gln Ala Ser 1 5 10

SEQ ID NO: 7 Sequence length: 13 Sequence type: Amino acid Topology: Linear Sequence type: Peptide sequence Ser Ala Gln Glu Lys Gln Asn Thr Glu Asn Glu Arg Leu 1 5 10

SEQ ID NO: 8 Sequence length: 13 Sequence type: Amino acid Topology: Linear Sequence type: Peptide sequence Thr Asn Gln Lys Glu Gln Ala Ser Leu Arg Glu Asn Glu 1 5 10

SEQ ID NO: 9 Sequence length: 13 Sequence type: Amino acid Topology: Linear Sequence type: Peptide sequence Thr Asn Gln Lys Glu Gln Ala Ser Glu Asn Glu Arg Leu 1 5 10

SEQ ID NO: 10 Sequence length: 26 Sequence type: Amino acid Topology: Linear Sequence type: Peptide sequence Gln Phe Leu Asn Glu Ser Trp Tyr His Arg Tyr Gln Asn Lys Arg Ala 1 5 10 15 Gly Ala Val Thr Asp Arg Asp Val Thr Gly 20 25

SEQ ID NO: 11 Sequence length: 21 Sequence type: Amino acid Topology: Linear Sequence type: Peptide sequence Glu Asn Glu Arg Leu Phe Phe Gly Ala His Ala Thr Ala Val Thr Asn 1 5 10 15 Gln Lys Glu Gln Gly 20

SEQ ID NO: 12 Sequence length: 23 Sequence type: Amino acid Topology: Linear Sequence type: Peptide sequence His Ala Thr Ala Val Thr Asn Gln Lys Glu Gln Ala Ser Leu His Val 1 5 10 15 Val Thr Gly Asn Leu Ala Leu Ser 20

SEQ ID NO: 13 Sequence length: 12 Sequence type: Amino acid Topology: Circular Sequence type: Peptide

SEQ ID NO: 14 Sequence length: 15 Sequence type: Amino acid Topology: Linear Sequence type: Peptide sequence Ala Lys Ala Trp Lys Lys Ala Ser His Leu Trp Lys Gly Ile Gly 1 5 10 15

SEQ ID NO: 15 Sequence length: 4 Sequence type: Amino acid Topology: Linear Sequence type: Peptide

SEQ ID NO: 16 Sequence length: 4 Sequence type: Amino acid Topology: Linear Sequence type: Peptide

SEQ ID NO: 17 Sequence length: 3 Sequence type: Amino acid Topology: Linear Sequence type: Peptide

[Brief description of drawings]

FIG. 1 is a graph showing the NGF production promoting activities of SEQ ID NOs: 10 and 55 kd TNF receptor-derived peptide (TVDRDTVAGARKNQYRHYWSENLFQG). The vertical axis represents the NGF production amount, and the horizontal axis represents the peptide concentration.

FIG. 2 NGF of peptides of SEQ ID NO: 2 and SEQ ID NO: 3
It is a graph which shows production promotion activity. The vertical axis represents the NGF production amount, and the horizontal axis represents the peptide concentration.

FIG. 3 is a graph showing the NGF production promoting activity of the peptide of SEQ ID NO: 4. The vertical axis represents the NGF production amount, and the horizontal axis represents the peptide concentration.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification code Office reference number FI Technical display location A61K 38/00 ADZ AED C07K 1/00 8318-4H 1/107 8318-4H 5/093 8318-4H 5/103 8318-4H 7/08 ZNA 8318-4H 14/48 8318-4H 14/525 8318-4H A61K 37/02 ADZ AED (72) Inventor British Tsunemoto 2-24-5 Midorigaoka, Zama, Kanagawa Prefecture ( 72) Inventor Risako Saida 2-22-2 Tamanawa, Kamakura City, Kanagawa Prefecture (72) Inventor Satoko Fuchida 2-3-24, Yokokodai, Isogo-ku, Yokohama City, Kanagawa Prefecture (72) Yukiko Hirota Nagasaki, Toshima-ku, Tokyo 4-31-4

Claims (11)

[Claims] 1. A whole amino acid sequence of a peptide or protein having 6 or more amino acid residues and having a useful biological activity or a partial amino acid sequence including a region expressing the activity, which constitutes non-overlapping parts of each other. An amino acid consisting of any two or more regions consisting of three or more amino acid residues, and any one or more or all of these regions in which the N-terminal side and the C-terminal side of the original amino acid sequence are reversed. A peptide or protein characterized by having a sequence and exhibiting an agonistic, antagonistic, or inhibitory activity of a peptide or protein having the above-mentioned useful biological activity (provided that it has an amino acid sequence that completely matches the reverse sequence of the original amino acid sequence). Peptides or proteins are excluded, and the amino group at the N-terminal of the peptide is acetyl or t-butoxyca. It may be modified with a bonyl group or a benzyloxycarbonyl group, and the C-terminal carboxyl group may be an amide group, and if two cysteines are present in the peptide, they are linked to form an intramolecular disulfide. May form a bond). However, when a cysteine residue is present in the amino acid sequence, the cysteine residue is glycine, alanine, proline, serine, threonine, phenylalanine, tyrosine, tryptophan, leucine, isoleucine, valine, methionine, lysine, arginine, Histidine, glutamine,
It may be substituted with an amino acid residue selected from the group consisting of glutamic acid, asparagine, aspartic acid, S-acetamidomethyl-cysteine, and α-aminoisobutyric acid. 2. The peptide or protein according to claim 1, which is represented by SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 4. 3. The peptide or protein according to claim 1, which is represented by SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12 or SEQ ID NO: 13 (provided that two Cys in the peptide of SEQ ID NO: 13 are bound to each other to form a molecule). Internal disulfide bond may be formed). 4. N-terminal side and C-terminal side of the entire amino acid sequence of a peptide or protein having a useful biological activity isolated from the natural world or an amino acid sequence consisting of amino acid residues of 3 residues or more and expressing said activity. Of the original peptide or protein having the above-mentioned useful biological activity, the peptide or protein having an amino acid sequence in which at least one amino acid residue is replaced with the same number of other amino acid residues. A peptide or protein having agonist, antagonist, or inhibitor activity (the amino group at the N-terminal of the peptide may be modified with an acetyl group, t-butoxycarbonyl group or benzyloxycarbonyl group, and the C-terminal carboxyl group is an amide group) And if two cysteines are present in the peptide, these May be taken together to form an intramolecular disulfide bond). However, the above-mentioned other amino acid residues are the amino acid residues to be replaced are group I (glycine, alanine, proline, serine, threonine), group II (phenylalanine, tyrosine, tryptophan), group III (leucine, isoleucine, valine). , Methionine), group IV (lysine, arginine, histidine) or group V (glutamine, glutamic acid, asparagine, aspartic acid), if any, the amino acid residue selected from the other amino acids in the group to which it belongs And the amino acid residue to be replaced is a cysteine residue, the above group I
To an amino acid residue selected from S-acetamidomethyl-cysteine and α-aminoisobutyric acid. 5. The peptide or protein according to claim 4, which is represented by SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16 or SEQ ID NO: 17 (however, , Two Cys in the peptide of SEQ ID NO: 13 may combine to form an intramolecular disulfide). 6. A whole amino acid sequence of a peptide or protein having 6 or more amino acid residues and having a useful biological activity, or a partial amino acid sequence containing a region expressing said activity, constituting a part which does not overlap with each other. Any two or more regions consisting of three or more amino acid residues; then consisting of two or more regions of which any one or more or all of the regions of each original amino acid sequence In the amino acid sequence in which the N-terminal side and the C-terminal side are reversed, or in the reversed amino acid sequence,
Synthesis of a peptide or protein derivative having an amino acid sequence in which at least one amino acid residue is replaced with the same number of other amino acid residues; and a useful peptide or protein characterized by evaluating its biological activity is searched for how to. However, the above-mentioned other amino acid residue means that the amino acid residue to be replaced is a group I (glycine, alanine,
Proline, serine, threonine), group II (phenylalanine, tyrosine, tryptophan), group I
II (leucine, isoleucine, valine, methionine),
If it belongs to either Group IV (lysine, arginine, histidine) or Group V (glutamine, glutamic acid, asparagine, aspartic acid), it is an amino acid residue selected from the other amino acids in the group to which it belongs, and When a cysteine residue is present in the amino acid sequence, the cysteine residue is glycine, alanine, proline, serine, threonine, phenylalanine, tyrosine, tryptophan, leucine, isoleucine, valine, methionine, lysine, arginine, histidine, It may be substituted with an amino acid residue selected from the group consisting of glutamine, glutamic acid, asparagine, aspartic acid, S-acetamidomethyl-cysteine, and α-aminoisobutyric acid. 7. The N-terminal side and C-terminal side of the entire amino acid sequence of a peptide or protein having a useful biological activity isolated from nature or an amino acid sequence consisting of three or more amino acid residues and exhibiting the activity. And a peptide or protein having an amino acid sequence in which at least one amino acid residue is replaced with the same number of other amino acid residues, and the biological activity thereof is evaluated. A method for searching for peptides or proteins. However, the above-mentioned other amino acid residues are group I (glycine, alanine, proline, serine, threonine), group II (phenylalanine, tyrosine, tryptophan), group III.
(Leucine, isoleucine, valine, methionine), group IV (lysine, arginine, histidine), or group V (glutamine, glutamic acid, asparagine, aspartic acid) from any other amino acid in the group to which it belongs When the amino acid residue to be selected is a cysteine residue, it is selected from any of the amino acids of Group I to Group V above, or S-acetamidomethyl-cysteine and α-aminoisobutyric acid. Amino acid residue. 8. SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4,
A tumor necrosis factor inhibitor comprising a peptide or protein represented by SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12 or SEQ ID NO: 13 or a physiologically acceptable salt thereof. 9. SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4,
Alternatively, a nerve growth factor production promoter comprising the peptide or protein represented by SEQ ID NO: 10 or a physiologically acceptable salt thereof. 10. A cell adhesion inhibitor comprising a peptide or protein represented by SEQ ID NO: 15, SEQ ID NO: 16 or SEQ ID NO: 17 or a physiologically acceptable salt thereof. 11. An antibacterial agent comprising a peptide or protein represented by SEQ ID NO: 14 or a physiologically acceptable salt thereof.