WO1994000488A1

WO1994000488A1 - Anti-hiv peptide or peptide derivative

Info

Publication number: WO1994000488A1
Application number: PCT/JP1993/000836
Authority: WO
Inventors: Kazuo Kumagai; Kazuyoshi Ikuta; Koji Ohki; Satoshi Mitsuda
Original assignee: Sumitomo Pharmaceuticals Company, Limited
Priority date: 1992-06-23
Filing date: 1993-06-22
Publication date: 1994-01-06

Abstract

A peptide or a peptide derivative represented by the following sequence (1), or a peptide or a peptide derivative wherein at least one amino acid in the sequence (1) is a D-amino acid, and a dimer thereof. In the sequence (1): R1-Asp Thr Ser Glu Asp Glu Val Ile Glu Lys Ile Leu Lys Asn Cys(R2) Lys Ile Gln Ile Glu Leu Asp Pro X Phe Glu Asn-R3, R1 represents hydrogen, a cysteine residue or an N-terminus-modifying group; R2 represents hydrogen or a cysteine-modifying group; R3 represents hydroxy, a cysteine residue or a C-terminus-modifying group; and X represents a tyrosine residue or a valine residue. The peptide or peptide derivative inhibits the infection with human immunodeficiency virus (HIV) and the formation of multinucleated giant cells after the infection with HIV, thus being usable for treating and preventing acquired immunodeficiency syndrome.

Description

Description Anti-HIV peptide or peptide derivative

Industrial applications

The present invention relates to anti-HIV peptide or peptide derivatives.

The anti-HIV peptide or peptide derivative of the present invention has a function of preventing infection of human immunodeficiency virus (hereinafter abbreviated as HIV), and further has an effect of forming multinucleated giant cells after HIV infection. It has an inhibitory function and is therefore used as a therapeutic or therapeutic agent for acquired immune deficiency syndrome (hereinafter abbreviated as AIDS). In addition, if its affinity for HIV is used, it can be used as a directional molecule to selectively act on ribosomes or proteinaceous cytotoxic substances that inhibit cellular protein synthesis on HIV-infected cells. .

Background of the Invention

A reverse transcriptase inhibitor such as azidothymidine (AZT) is used clinically as a drug for treating or preventing the development of AIDS, but it has problems such as side effects on bone marrow and the emergence of resistant HIV strains It has been.

Therefore, in order to develop drugs with high efficacy and low toxicity, new drugs that inhibit the infection of cells with HIV are being developed. One such is a sulfated polysaccharide such as dextran sulfate. However, sulfated polysaccharides are said to have problems such as low absorbability when administered orally and short blood half-life even when administered intravenously.

On the other hand, attempts are being made to use soluble CD4 derived from human CD4 or its fragment peptides. CD4 is the surface of cells such as helper T lymphocytes Is a membrane glycoprotein with a molecular weight of about 55 kilodaltons and is a cell-side receptor for HIV infection. Soluble CD4 is a protein (glycoprotein) composed of 370 amino acid residues outside the cells of the CD4 molecule, and is known to exhibit anti-HIV activity (DH Smith et al.). al, .Science. 238. 1704—1707, 1987). However, since soluble CD4 is a large protein molecule, it usually has to be produced by genetically engineered recombinants, and there is a problem that it is expensive and time-consuming to provide a sufficient amount. . On the other hand, CD4 fragment peptides derived from the partial structure of CD4 can be chemically synthesized as long as they are several tens of amino acid residues long, and their development as anti-HIV agents has been studied. (JP-A-2-131497, JP-A-2-152989, J. D. Lifson et al,. Scienc e. 241. 712-716. 1988). In particular, peptides corresponding to the N-terminal amino acid residues 66 to 92 of CD4 (CD4 [66-92]) or derivatives thereof can strongly suppress HIV infection to cells. It has been clarified (JP-A-3-38599).

—On the other hand, since HIV is produced by cells infected with HIV, the development of a therapeutic agent for AIDS using these HIV-infected cells as an action point is also being studied. For example, attempts have been made to selectively kill HIV-infected cells by binding a protein fragment of ricin toxin or Pseudomonas aeruginosa exotoxin A to soluble CD4 (M. A, Ti 11 et al, Science). .242. 1166-1168, 1988: VK Cha udhary et al,. Nature. 335. 369-372, 1988). On the other hand, liposome-based drugs are also being studied, and liposomes consisting of phosphatidylcholine, which has a phase transition temperature of less than 37 ° C, and acidic phospholipids and cholesterol, can selectively kill HIV-infected cells. It has been clarified (Japanese Patent Laid-Open Publication No. 3-236325).

-- However, although various drugs are being studied as described above, further development of new drugs is required in view of efficacy, toxicity, or emergence of resistant HIV strains. Summary of the Invention

The present inventors have conducted intensive studies to solve the above-mentioned problems, and as a result, at least one of the peptide or peptide derivative represented by the following sequence 1 or the amino acid residue of the sequence 1 is a D-amino acid. A peptide or peptide derivative that is:

Array 1-

Ri-Asp Thr Ser Glu Asp Glu Val lie Glu Lys lie Leu

5 1 0

Lys Asn Cys (R ₂ ) Lys lie Gin lie Glu Leu Asp Pro X

1 5 20

Phe Glu Asn-Rs

twenty five

(Wherein, R, represents a hydrogen atom, a cysteine residue or a N-terminal modifying group, R ₂ represents a hydrogen atom or a cysteine modifying group, R ₃ represents a hydroxyl group, a cysteine residue or a C-terminal modifying group, X Represents a tyrosine or valine residue), or

A peptide or a peptide derivative represented by the following sequence 2 or 3 or a peptide or a peptide derivative in which at least one of the amino acid residues of the sequence 2 or 3 is 0-amino acid: Array 2

R ₄ -Asp Thr Ser Glu Asp Glu Val lie Glu Lys lie Leu Lys Asn Cys (R ₂ ) Lys lie Gin lie Glu Leu Asp Pro X Phe Glu Asn- Y

R ₄ -Asp Thr Ser Glu Asp Glu Val lie Glu Lys lie Leu Lys Asn Cys (R ₂ ) Lys lie Gin lie Glu Leu Asp Pro X

Phe Glu Asn- Y

, Or

Array 3

Y- Asp Thr Ser Glu Asp Glu VaL lie Glu Lys lie Leu Lys Asn Cys (R ₂ ) Lys lie Gin lie Glu Leu Asp Pro X Phe Glu Asn— R ₅

(In the formula, R ₄ represents a hydrogen atom or a N-terminal modification group, R ₂ represents a hydrogen atom or a cysteine modification group, R ₅ represents a hydroxyl group or a C-terminal modification group, and X represents a tyrosine residue or palline. Represents a residue. One Y—Y— represents one Cys—S—S—Cys— or a bifunctional crosslinking reagent.

However, they have found that they have a strong anti-HIV action, and have completed the present invention.

Since the anti-HIV peptide or the peptide derivative of the present invention has a very strong antiviral effect that strongly inhibits both HIV infection of CD4-positive cells and intercellular infection involving multinucleated giant cell formation, Useful as an anti-HIV agent. The peptide or peptide derivative of the present invention is HI It has been found that the ribosome to which the peptide or the peptide derivative of the present invention is bound has an excellent selective killing effect on HIV-infected cells. Therefore, they can be used as a therapeutic or preventive for AIDS. BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a graph showing the CD spectrum of peptide 1 (solid line) and peptide 4 (dashed line).

FIG. 2 shows the activity of the anti-HIλ peptide (peptides 1, 4, 5, and 6) of the present invention to inhibit infection of HIVCHTLV-ICB) to CD4 positive cells (M10).

FIG. 3 is a graph comparing the selectivity of a liposome bound with the anti-HIV peptide of the present invention (peptide 1) to HIV-infected cells and non-infected cells. Closed circles indicate the growth inhibition rate against HIV infected cells (MOLT-4ZLAV-1), and open circles indicate the growth inhibition rate against uninfected cells (MOLT-4) (cultured at 37 ° C for 3 days).

FIG. 4 is a graph comparing the selectivity of a liposome bound with an anti-HIV peptide (peptide 2) of the present invention to HIV-infected cells and non-infected cells. Closed circles indicate the growth inhibition rate against HIV infected cells (MOLT-4AV-1), and open circles indicate the growth inhibition rate against uninfected cells (MOLT-4) (culture at 37 ° C, .3).

FIG. 5 is a graph comparing the selectivity of ribosomes to which the anti-HIV peptide of the present invention is not bound to HIV-infected cells and non-infected cells. Closed circles indicate growth inhibition rate against HIV-infected cells (MOLT-4 LAV-1), and open circles indicate growth inhibition rate against uninfected cells (MOLT-4). (37 ° C, 3 culture). Detailed description of the invention

The present invention provides an anti-HIV peptide or a peptide derivative that inhibits the transmission of HIV infection. .

The present invention also relates to an anti-HIV peptide-bound ribosome or a protein that inhibits cell protein synthesis, which significantly enhances selective killing of HIV-infected cells by binding to the anti-HIV peptide or peptide derivative. Toxic cytotoxic substances.

The configuration of each amino acid residue in the anti-HIV peptide or peptide derivative of the present invention represents the L-form unless otherwise specified.

The anti-HIV peptide or peptide derivative of the present invention also includes those in which at least one of the respective amino acid residues is substituted with an amino acid residue having a D configuration.

In the above formula, “bifunctional cross-linking reagent (or cross-linking agent)” includes in-maleimidobenzoinole N-hydroxysuccinimido-estenole (m-maleimidobenzoyl-N-hydroxysuccinimide ester, MBS). during ₀ formula that, as the "N-terminal modifying group", for example, various amino acid residues of various Bae flop tides, alkyl group, Ashiru group, a substituted alkyl group, and substituted Ashiru group like et be.

Examples of the amino acid residues include alanine, arginine, asparagine, asparaginic acid, cystine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, and the like. Residues such as serine, threonine, tryptophan, tyrosine, valin and the like can be mentioned. Examples of the peptides include oligopeptides and polypeptides composed of the above amino acid residues.

Examples of the alkyl group include a linear or branched lower alkyl group having 6 or less carbon atoms, and specifically, methyl, ethyl, propyl, 2-propyl, butyl, 2-butyl, 3-butyl, Examples thereof include 1,1-dimethylethyl, pentyl, and hexyl.

Examples of the acyl group include an alkanoyl group and an aroyl group.

Examples of the alkanoyl group include a lower alkanoyl group having 7 or less carbon atoms. Examples of the lower alkanoyl group include a formyl group, an acetyl group, a propanoyl group, a butanol group, a pentanoyl group, a hexanoyl group, and a heptanyl group. Examples of the aryloyl group include a lower aryloyl group having 7 or less carbon atoms. The lower arylo group includes, for example, a benzoyl group.

Examples of the substituent of the substituted alkyl group include a halogen atom such as a fluorine atom, a chlorine atom, a bromine atom and an iodine atom, an alkoxy group having 4 or less carbon atoms such as methoxy, ethoxy, propoxy, 1-methylethoxy, and an amino group. And aromatic groups such as lower alkylamino group, di-lower alkylamino group and phenyl group, and substituted aromatic groups such as substituted phenyl group. Examples of the substituted aromatic substituent include a halogen atom such as a fluorine atom, a chlorine atom, a bromine atom and an iodine atom, an alkoxy group having 4 or less carbon atoms such as methoxy, ethoxy, propoxy and 1-methylethoxy, and an amino group. And lower alkylamino groups and di-lower alkylamino groups.

Examples of the substituent of the substituted ethyl group include methyl, ethyl, propyl, 2-propyl, butyl, 2-butyl, 3-butyl, 1,1-dimethylethyl, pentyl, and the like having 6 or less carbon atoms. Lower alkyl group such as hexyl, fluorine source Halogen atoms such as chlorine atom, bromine atom and iodine atom, alkoxy groups having 4 or less carbon atoms such as methoxy, ethoxy, propoxy, and 1-methylethoxy, amino groups, lower alkylamino groups, and di-lower alkyl Examples include an amino group.

In the formula, examples of the “C-terminal modifying group” include various amino acid residues, various peptides, alkyl groups, substituted alkyl groups, amino groups, lower alkylamino groups, di-lower alkylamino groups, and the like.

Examples of amino acid residues include, for example, alanine, arginine, asparagine, aspartic acid, cystine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine-, methionine, phenylalanine, proline, and serine. , Threonine, tryptophan, tyrosine, valin and the like.

Examples of the peptides include oligopeptides and polypeptides composed of the above amino acid residues.

Examples of the lower alkyl group include straight-chain or branched groups having 6 or less carbon atoms, and specifically, methyl, ethyl, propyl, 2-propyl, petyl, 2-butyl, 3-butyl, 1, 1-dimethyl-ethyl, pentyl, hexyl and the like.

Examples of the substituent of the substituted alkyl group include a halogen atom such as a fluorine atom, a chlorine atom, a bromine atom and an iodine atom, an alkoxy group having 4 or less carbon atoms such as methoxy, ethoxy, propoxy, and 1-methylethoxy, an amino group, Examples include aromatic groups such as a lower alkylamino group, a di-lower alkylamino group, and a fuunyl group, and substituted aromatic groups such as a substituted phenyl group. Examples of the substituted aromatic substituent include methyl, ethyl, propyl, 2-propyl, butyl, 2-butyl, 3-butyl, 1,1-dimethylethyl, pentyl, and the like having 6 or less carbon atoms. A lower alkyl group such as hexyl, a halogen atom such as a fluorine atom, a chlorine atom, a bromine atom, and an iodine atom; an alkoxy group having 4 or less carbon atoms such as methoxy, ethoxy, propoxy, and 1-methylethoxy; an amino group; Examples thereof include an alkylamino group and a di-lower alkylamino group.

Examples of the “cysteine modifying group” include an alkyl group, a substituted alkyl group, an acetamido lower alkyl group, and a lower alkyl group rubamoyl group.

Examples of the substituent of the substituted alkyl group include a halogen atom such as a fluorine atom, a chlorine atom, a bromine atom and an iodine atom, an alkoxy group having 4 or less carbon atoms such as methoxy, ethoxy, propoxy, 1-methylethoxy, and an amino group. And aromatic groups such as lower alkylamino group, di-lower alkylamino group and phenyl group, and substituted aromatic groups such as substituted phenyl group. Examples of the substituted aromatic substituent include methyl, ethyl, propyl, 2-propyl, butyl, 2-butyl, 3-butyl, 1,1-dimethyl-ethyl, pentyl, and hexyl having 6 or less carbon atoms. A lower alkyl group, a fluorine atom, a chlorine atom, a bromine atom, a halogen atom such as an iodine atom, an alkoxy group having 4 or less carbon atoms such as methoxy, ethoxyquin, propoxy, 1-methylethoxy, an amino group, a lower alkylamino group, And di-lower alkylamino groups.

As a method for producing the anti-HIV peptide of the present invention, a peptide synthesis method by a solid phase method is suitable. As a peptide synthesis method by the solid phase method, a method using Fmoc amino acid (E. Atherton and Ε · C. Sheppard, J. Chem. Soc. Che. m. Connnun., 165-166.1985). That is, a resin obtained by adding 9-fluorenylmethyloxycarbonyl (Fmoc) amino acid residue corresponding to the C-terminus of a peptide to a methylbenzhydrylamine polystyrene resin using hydroxymethylphenoxyacetic acid as a linker. Then, the Fmoc amino acid residue is sequentially bonded by a condensation reaction. After binding each amino acid residue, the resin is treated with a mixture of 30% piperidine and dimethylformamide to remove the Fmoc group at the N-terminal of the peptide, and then treated with TFA in the presence of m-cresol and ethanedithiol at room temperature. Deprotection is performed by the action of a mixture of (trifluoroacetic acid) and monothioisole, and the desired peptide is obtained.

Synthesis of Derivatives at Cysteine Residues · To synthesize peptides, which are benzylated products at thiol groups, Fmoc—Cys (B ^ l) It can be synthesized by using [9-fluorenylmethyloxycarbonyl-S-benzylsilstein]. The benzyl group is not cleaved in the presence of m-cresol and ethanedithiol by the protective group excision operation at room temperature using a mixture of TFA and thioanisole, so that a peptide in which the cysteine residue is derivatized with the benzyl group is obtained. . On the other hand, in order to synthesize a peptide in which the thiol group of the cysteine residue is in a free form, Fmoc—Cys (Trt) [9-fluorenylmethylquinoline carbon— Trityl cysteine] is preferably used. The trityl group can be formed together with other protecting groups by a normal protecting group excision operation by treating in a mixture of TFA-thioanisole (for example, 95: 5) for 1 hour at room temperature in the presence of m-cresol and benzodithiol. Since the peptide is excised, a peptide in which the thiol group of the Cys residue is free can be obtained.

The obtained peptide is used for chromatography such as high performance liquid chromatography (HPLC). It can be purified by chromatography. In addition, it can also be produced by a method using other peptide synthesis methods, or a method of preparing DNA corresponding to the peptide, connecting it to an appropriate vector, and expressing it in animal cells or microorganisms. it can.

In another embodiment, the anti-HIV peptide or peptide derivative of the present invention can be used as a directional component for selectively causing a proteinaceous cytotoxic substance that inhibits cell protein synthesis to act on HIV-infected cells. It can also be used for liposomes. As a method for binding a peptide to a liposome or a cytotoxic substance, a method known in the production of immunoribosome or imnotoxin (for example, -Methods in Enzymology, 149.111-119 987) Can be used. When binding to ribosomes, a thiol group can be added to the peptide using a bifunctional cross-linking reagent such as N-succinimidyl 3- (2-pyridylthio) propionate (N-succin imidyl 3- (2-pyridyldithio) propionate: SPDP). After introducing the liposome, a peptide-phospholipid conjugate is prepared by reacting with a phospholipid having a linking group capable of reacting with a thiol group. Make up one song. Examples of the phospholipid having a binding group capable of reacting with a thiol group include pyridyldithiopropionylphosphatidylethanolamine obtained by reacting phosphatidylethanolamine with SPDP. Examples of the phosphatidylethanolamine used in the above include phosphatidylethanolamine of animal origin, and phosphatidyl obtained by substituting an acyl group with a lauroyl group, a myristoyl group, a palmitoyl group, a stearoyl group, an oleoyl group, or the like. Ethanolamine is mentioned, and phosphatidylethanolamine of animal origin is particularly preferable. On the other hand, thiol groups derived from cysteine residues are present in the peptide In this case, instead of introducing a thiol group into the peptide using the above bifunctional crosslinking reagent, the thiol group in the peptide can be used as it is.

As the lipid composition of the ribosome to be used, various compositions can be used as long as the ribosome is formed, but a particularly preferred composition has a phase transition temperature.

37. The above-mentioned conjugate of beptidophospholipids in a composition in which phosphatidylcholine having a temperature of less than 77 ° C., acidic phospholipids and cholesterol are preferably in a molar ratio of 5: 1: 2 is 0.1 to 10 mol%. Added compositions are included. Phosphatidylcholine having a phase transition temperature of less than 37 ° C includes animal and plant lecithin (eg, egg yolk lecithin, soybean lecithin), and the substitution of an acyl group with a lauroyl, myristoyl, or oleoyl group. Phosphatidylcholine is mentioned, and a particularly preferred example is dimyristoylphosphatidylcholine (phase transition temperature: 23 ° C.). On the other hand, acidic phospholipids include animal-derived phosphatidylserine, phosphatidylglycerol, phosphatidylinositol, phosphatidic acid, and acylic acid in the form of lauroyl, myristyl, panolemityl, and stearoyl groups. Examples thereof include phosphatidylserine, phosphatidylglycerol, phosphatidylinositol, and phosphatidic acid substituted with an oleoyl group and the like. Particularly preferred examples include phosphatidylglycerol derived from animals.

As a method for producing liposomes from these liposome membrane components, known techniques are used. As the production method,

(1) After mixing the components of the liposome membrane in a solvent, the solvent is removed by evaporation to form a lipid thin film, which is hydrated by adding a buffer solution, and then shaken or stirred to produce a multilamellar liposome (MLV). How to get the (2) a method of obtaining liposomes having a uniform particle size by filtering MLV through a membrane filter,

(3) A method for obtaining small single membrane ribosomes (SUV) by treating MLV with an ultrasonic crusher,

And the like. Any buffer can be used as long as it can be used for the production of ribosomes, and examples thereof include a phosphate buffer, a citrate buffer, a lactate buffer, and an acetate buffer. The pH of the buffer is preferably about pH 6 to about neutral, and the concentration of the buffer is preferably about 5 to 5 OmM. These buffers may also be made isotonic by adding salt, glucose, sucrose, or the like. Further, physiological saline may be used in place of the buffer.

Liposomes composed of phosphatidylcholine, which has a phase transition temperature of less than 37 ° C, acidic phospholipids, and cholesterol are known to act killer on HIV-infected cells ^ — 2 3 6 3 2 5), the ribosome of the present invention in which an anti-HIV peptide is bound to the ribosome has higher selectivity for HIV-infected cells, and kills and destroys HIV-infected cells selectively and effectively. Because it breaks down, it is useful as an AIDS treatment or onset prevention agent.

The number of peptides that can be bound to the liposome membrane can theoretically be up to about 800 per liposome, but 10 to 10 0 is preferred.

In addition, the formed liposome can hold a drug in its inner aqueous layer, and is therefore useful as a drug delivery body. As the drug to be retained, a toxin (for example, diphtheria toxin fragment A) that inhibits protein synthesis of cells and an anti-HIV agent such as AZT are suitable. Some HIV-infected cells have a phagocytic effect (for example, macrophages infected with HIV), so the cellular protein synthesis A liposome carrying a toxin that inhibits these is thought to be useful for killing these cells.

On the other hand, by binding the anti-HIV peptide or peptide derivative of the present invention to a proteinaceous cytotoxic substance that inhibits cell protein synthesis, the cytotoxic substance can be selectively transmitted to HIV-infected cells. And can selectively kill the infected cells. Examples of proteinaceous cytotoxic substances that inhibit cellular protein synthesis include ricin, gelonin, diphtheria toxin, Pseudomonas aeruginosa exotoxin A, and toxicologically active fragments thereof. In order to bind the anti-HIV peptide of the present invention to the cytotoxic substance, a thiol group is introduced into both the peptide and the cytotoxic substance using the aforementioned bifunctional crosslinking reagent such as SPDP. Are linked by a disulfide bond. However, when a free thiol group is present in the molecule of the anti-HIV peptide or the cytotoxic substance, the operation of introducing the thiol group using the above bifunctional crosslinking reagent can be omitted.

On the other hand, the anti-HIV peptide or peptide derivative of the present invention can also be produced as a single fusion protein by linking it to a cytotoxic substance via a peptide bond. In such a case, a method is used in which DNA corresponding to the fusion protein is prepared, ligated to an appropriate vector, and expressed in animal cells or microorganisms for production. The peptide substance of the present invention in which the anti-HIV peptide is bound to a cytotoxic substance exhibits selective damage to HIV-infected cells, and is therefore used as a therapeutic agent for AIDS and a preventive agent for the development of AIDS.

The anti-HIV peptide or the peptide derivative of the present invention, the ribosome to which the anti-HIV peptide is bound, or the peptide substance obtained by combining the anti-HIV peptide and the cytotoxic substance are used alone or in combination. When used as a therapeutic or preventive agent for the disease, the dosage An amount is used, for example, of 0.01 to 1 Omg as the inventive peptide or peptide derivative per kg body weight by intravenous administration or infusion. Example

Next, examples of the present invention will be described. However, these examples are merely examples, and do not limit the present invention. In this example, when no distinction is made between amino acid residues in D-form and L-form, it is assumed that the amino acid residue is in the L-form.

Example 1

Asp-Thr- Ser-Glu- Asp-Glu- Val- I le-Glu-Lvs- I le One Leu— Lys— Asn— Cys (Bzl) — Lys— I le— Gin- I le— Glu— Leu — Asp — Pro— Tyr—Phe— Glu— Asn (Peptide 1) Preparation

F moc—A sn (T mob) PAC ^™ resin (ie, N.19-Fluorenylmethyloxycarbonyl N 2,4,6-trimethoxybenzyl-L-as paragine PAC ^™ resin) [Miridinnobio (O. lmmole) as Fmoc-amino acid residues as Fmoc-Val-Fmoc-Ile-Fmoc-Leu-Fmoc-Asn (Tmob) -Fmoc-Asp (OtBu), Fmoc-Glu (〇tBu) , Fmoc-Thr (tBu) ^ Fmoc- Ser (tBu), Fmoc- Lys (Boc), Fmoc- Cys (B zl), Fmoc- Gin (T mob), Fmoc- Phe, Fmoc-Pro, Fmoc- Tyr Each residue of (tBu) (0.5mmole each) (here, Tmob means 2,4,6-trimethoxybenzyl, tBu means tert-butyl), and in dimethyl formamide (DMF) , 1-hydroxybenzotriazole (0.5ππηο1e), benzotriazolyloxytrisdimethylaminophosphoniumhexafluorophosphate (O. Snunole) and add Extension of the tide chain was performed. These series of reactions were performed using a peptide synthesizer manufactured by Miridyun / Biosearch.

(EXCELL ^™ ). After the completion of all the coupling reactions, the N-terminal Fmoc group was removed with 30% piperidine-DMF, and then the protecting group of each amino acid residue except Cys (Bzl) residue was removed and the resin was removed from the resin. To excise the peptide, it was treated in trifluoroacetic acid (TFA) -thioazole in the presence of m-cresol and ethanedithiol for 1 hour at room temperature. Then, the mixture was filtered through a glass filter, the filtrate was concentrated, and ether was added thereto to obtain a white peptide powder. This was dissolved in a mixture 51 ^ of 2-mercaptoethanol / acetic acid / water = 255, applied to a Sephadex G-15 (Pharmacia) column, and eluted with 50% acetic acid to remove low molecular impurities. This was further purified by reverse phase HPLC. The conditions for preparative purification by HPLC are as follows: Column: YMC Pack SH—363-5 (3 Omm0 × 250 thigh), eluent A: 0.1% TFA, eluate B _: acetonitrile containing 0.1% TFA, gradient: A Solution: Solution B 80%: 20% → 60%: 40% (100 minutes), Flow rate: 9.9m ^ / min, Detection: UV absorption at 22 Onm. The target peptide was eluted with a retention time of 88 minutes. The purity of the obtained peptide (20 ^) was analyzed by HPLC under the following conditions. Column: YMC Pack AM-303 (4.6 mm x 250 mm), Eluent A: 0.1% TFA, Eluent B: acetonitrile containing 0.1% TFA, gradient; A solution 100% → A solution: A solution 40% : 60% (60 min), Flow rate: 1. Oral, min, Detection: UV absorption at 22 Onm. The peptide of interest eluted as a single peak with a retention time of 47 minutes. The amino acid sequence analysis (Edman degradation method) and the molecular weight analysis (FAB-Mass method) of the obtained peptides were all in agreement with the theoretical values. Example 2

Asp- Thr- Ser-Glu- Asp-Glu- Val- I le-Glu-Lys- I le — Leu— Lys— Asn— Cys (Bzl) — L vs— I le— Gin— I le— Glu— Leu

— 513—? ]: 0 d 31-? Preparation of 1½-011] _ ⁵¹¹ (peptide 2)

In Example 1, only the Fmoc-Tyr (tBu) residue was used as the Fmoc-amino acid residue in Example 1 and the other methods were the same as in Example 1 to synthesize the peptide. Purification and analysis yielded 18 mg of the desired peptide.

Comparative Example 1

I le-Lys-Glu- Cvs (Bzl)-Val- Asn- Asp- Ser- I le— Glu

Q

-Preparation of Gin-Lys-Glu-Pro-Leu-Asp-Tyr-Ile-Glu-Leu-Phe-Glu-Lys-Thr-Asp-Ile-Asn (Peptide> Q3)

The peptide was synthesized, purified and analyzed in the same manner as in Example 1 except that the order of the Fmocamino acid residues to be bound was changed. The peptide had the same amino acid composition as Peptide-1, but only the sequence order was the same. 18 ^ different peptide compounds (peptide 3) were obtained.

Example 3

D- Asp-D-Thr-D- Ser-DG lu-D- Asp- D-Glu-D- Val-D- I le— D— Glu— D— Lys— D— I le- D-Leu- D-Lys-

D—Asn—L-Cys (Bzl) —D—Lys—D—Gln-D-I le—

Preparation of D-Glu—D—Leu—D—Asp—D—Pro—D-Phe-D-Glu—L—Asn (Peptide 4)

In Example 1, Fmoc-D-Val as a Fmoc amino acid residues, Fmoc-D-l ieヽ_{Fmoc- D- Leu s Fmoc- D- Asn (} l mobA Fmoc- D- A sp (OtBu), Fmoc- D — Glu (OtBu), Fmoc— D— Thr (tBu), Fmoc One D—Ser (tBu), Fmoc—D—Lys (Boc), Fmoc—L One Cys (Bzl), F moc—D—Gln (Tmob), Fmoc—D—Phe, Fmoc—D—Pro, Fmoc— The peptide was synthesized, purified and analyzed in the same manner as in Example 1 except that each residue of D-Tyr (tBu) was used, and the amino acid sequence was the same as that of peptide-1. peptides (peptide - 4) residues in Cys (Bzl) and C the remaining amino acid residues except the Asn terminal all _D-c then obtain 10 m ₉ a, circular dichroism for the peptide (CD) The spectrum was measured. The measurement conditions were as follows: measuring device: Jasco J-720 (manufactured by JASCO Corporation), solvent: 10 mM sodium phosphate buffer (pH 7.2), peptide concentration: 0.

Cell optical path length: 0.2cm, measurement wavelength range: 190-300nm, number of integration: 5 times, measurement temperature: 37 ° C. Fig. 1 shows the obtained CD spectrum. In FIG. 1, the dashed line shows the CD spectrum of the peptide of the present invention (peptide 14), and the solid line shows the CD spectrum of peptide 1 of the same amino acid sequence as the present peptide and all amino acid residues are in L form. Represents a vector. Both peptides showed diametrically opposite circular dichroism, indicating that the present peptide (peptide 4) retains the configuration of D-form amino acid residues even in aqueous solution.

Example 4

Preparation of dimeric peptides (peptides 5 and 6) by introduction of Cys residue into peptide 1

In Example 1, in addition to those used in Example 1 as Fmoc amino acid residues, Fmoc-Cys (Trt) residues were used for adding a Cys residue to the N-terminus. The synthesis, purification and analysis of the peptide were performed in the same manner as described above. This allows the amino acid sequence to be

Cys- Asp-Thr- Ser-Glu- Asp- Glu- Val- Ile— Glu— Lys- Ile- Leu- Lys- Asn- Cys (Bzl)-Lys- Ile- Gin- Ile— Glu— Leu- Asp- Pro-Tyr- Phe-Glu- Asn

1 peptide was obtained. This was dissolved in 5 5OmM sodium phosphate buffer solution (pH 7.2), and the N-terminal Cys residue was air-oxidized between the peptide molecules by gentle stirring at room temperature for 6 hours. The resulting dimeric peptide was purified under the HPLC conditions described in Example 1 to obtain a peptide (peptide) in which dimerized peptide-1 was obtained via a Cys residue added to the N-terminus. 5) got 5 ^.

In Example 1, a Fnioc-Cys (Trt) -Alko (Wang) resin (manufactured by Watanabe Chemical Industry Co., Ltd.) was used in place of the Fmoc-Asn (Tmob) PAC ^™ resin. Using a 90δ0 type peptide synthesizer manufactured by Milliyun Nova Search Co., Ltd., the other steps were the same as in Example 1 to synthesize, purify, and analyze the amino acid sequence. Asp— Thr— Ser— Glu— Asp— Glu— Val— I le— Glu— Lys— I le— L eu— Lys— A sn— Cys (B z 1) — Lys— I le— Gin— There were obtained 15 peptides designated as Ile-Glu-Leu-Asp-Pro-Tyr-Phe-Glu-Asn-Cys. This was dissolved in 7.5m of 5OmM sodium phosphate buffer (pH 8.0) and gently stirred at room temperature for 24 hours to air-oxidize the C-terminal Cys residue between the peptide molecules. The resulting dimeric peptide was purified under the HPLC conditions described in Example 1 so that the peptide (peptide 6) was obtained by dimerizing peptide 1 via the Cys residue added to the C-terminus. ) Was obtained 5 ^.

Example 5

Anti-HIV activity of the peptides of the present invention

The culture supernatant of MOLT-4 cells, which are persistently infected with HIV (HTL V-II), is used as the HIV solution. After serially diluting the HIV solution (0.05 ml), the synthesized peptides are shown in the following table. Add 0.05 ml at various final concentrations shown in 1 and 37 ° C For 30 minutes. Next, each mixture was combined with HIV-sensitive MT-14 cells (1 X

And 1: 1 (v / v) (total volume: 0.2 m), and cultured in a 5% carbon dioxide incubator at 37 ° C. for 4 days (quadruplicate). The culture medium used was RPM1-1640 medium supplemented with 10% fetal bovine serum. After completion of the culture, the infection titer (TCID5 ₍₎ Zm was measured by immunofluorescence using a monoclonal antibody against the HIV gag protein pi7, and the peptide's activity to inhibit infection was determined.

On the other hand, in the cell suspension of MOLT-4 cells, which are persistently infected with HIV (HTLV-ΠIB), 0.05 ^^ (5 × 10 ³ cells) of each of the synthesized peptides were synthesized as shown in Table 1 below. At a final concentration of 0.05 ml and kept at 37 ° C for 30 minutes. Then, each of the resulting mixtures is mixed with 0.1 m (5 x 10 ⁴ cells) of a cell suspension of uninfected MOLT-4 cells. The cells were cultured in a 5% carbon dioxide incubator at 37 ° C for 24 hours (triplicate). After completion of the culture, the number of multinucleated giant cells formed by cell-to-cell infection was measured by microscopic observation, and the inhibitory activity of the peptide on multinucleated giant cell formation was determined.

The results obtained are shown in Table 1 below. Peptide 1 and Beptide 2 showed very strong activity of inhibiting HIV infection and formation of multinucleated giant cells. In contrast, Peptide-3, a control, showed little activity against both HIV infection and multinucleated giant cell formation. The above results indicate that peptide-3, which has the same amino acid composition as peptide 1 and differs only in sequence, has no anti-HIV activity. The anti-HIV activity of the anti-HIV peptide of the present invention is similar to that of amino acid. This indicates that the specificity depends on the sequence of the residues.

Next, the anti-HIV activities of Peptide-4, Peptide-5, and Peptide-16 were examined in the same manner. Table 2 shows the results. All three peptides Very strong anti-HIV activity was observed. In particular, dimerized peptides of peptide-1 such as heptatide 5 and heptatide-6 showed extremely strong anti-HIV activity, including activity at a concentration of 4 g / nLg. Furthermore, HIV (HTLV-mB) under peptide presence mentioned various concentrations ^{_{(2x l 0 3 TC ID 5}} .) And 4 days at 37 ° C for with Ml 0 cells are CD 4-positive cells (10 ⁴ cells) After culturing, the percentage of cells positive for HIV antigen at that time was examined by the immunofluorescence method described above. The results obtained are shown in FIG. As a result, it was recognized that HIV antigen expression was suppressed depending on the peptide concentration. The above results indicate that the anti-HIV peptide of the present invention (peptide 1) strongly inhibits HIV infection of CD4-positive cells and intercellular infection involving multinucleated giant cell formation. It has been shown that the anti-HIV effect is maintained even when the amino acid residue is in the D-form, and that the effect can be further enhanced by dimerization. Peptide concentration (MgZm infectivity titer (TCID ₅ ./nLi) multinucleated giant cell formation rate (%) No addition 10 ^6. ⁰ 100

Bae Puchido 1 1 10 ² ^2.0 4.9

0.5 10 ³ ^2.0 14.8

0.25 10 ³ - ⁰ 45.2

0.125 10 ⁴ - ⁵ 82.7 Bae Puchido 2 1 10 ^{^2-5} 6.7 Bae Puchido 3 1 10 ^{^5.5} 85.9

0.5 0.5 10 ⁵

^{^{0. 2 δ 1 ο 6 · 0}} 96. 7

^{^{- 0. 125 1 ο 6 · 0}} 97. 6 Table 2

Peptide concentration (/ in Infectious titer (TCID _{5 ()} / m Multinucleated giant cell formation rate (%) No addition 1 0 ⁵ · ⁵ 100

Bae Puchido 4 1 <1 0 ² ^2.0 5.1

0.25 1 0 ³ - ⁰ 29.9

0.063 1 0 ⁴ - ⁰ 49.0

0.016 1 0 ⁵ - ⁰ 69.0

0.004 2

2 1 0 ⁵ ⁵ 89.9 Peptide 0 1 <1 0 ²

^{^{0. 25 <1 ο 2 · 0}} 7. 4

^{^{0. 063 1 ο 2 · 5 18.}} 2

^{^{0. 016 1 ο 4 · 0 32.}} 2

0. 004 1 ο ⁵ · ⁰ 51. 0 Peptide 6 1 <1 ο ² · ⁰ 1.6

^{0. 25 <1 Ο '2 ·} 0 2. 2

0.063 10 ² - ³³ 18.3

^{^{0. 016 1 ο 3 · 5 33.}} 0

^{^{0. 004 1 ο 4 · 5 59.}} 0

Example 6

Measurement of cytotoxicity of the peptide of the present invention

Various concentrations of peptides were added to the cells used in Example 5, and the cells were cultured at 37 ° C. for 3 days with the initial number of cells of ⁵ × 10 ⁵ cells. After cultivation, transfer 500 1 of the culture solution to a 96-well plate, and add 401 medium (RPM I-1640 medium supplemented with 10% fetal bovine serum) and 101 thiazolyl blue tetrazolium umb. MTT) solution (dissolved in phosphate buffered saline at a concentration of 5 and filtered and sterilized), and incubated at 37 ° C for 3 hours. Next, 100 1 of a 0.1% hydrochloric acid solution of 10% sodium dodecyl sulfate was added, and the mixture was incubated at 37 ° C. for 24 hours. The generated blue dye (formazan) was quantified by an absorbance of 57Οηπι. Based on the proportionality between the amount of formazan, which is a biodegradation product of MTT, and the number of viable cells, the ratio of the number of viable cells was determined from the above absorbance value. Table 3 shows the results. None of the peptides showed any cytotoxicity in the concentration range examined.

Table 3

Number of viable cells (%) (percentage of peptide not added) 濃度 Peptide concentration (m _ff no! N £) ΜΤ- ■ 4 MOLT-4 ぺ Peptide-1 1 98. 7 99 2

0 2 δ 1 0 1.2 1 02.3

0.0 6 3 9 7.6 1 0 δ.0 peptide -2 1 1 02. 0 9 7.6

0.25 1 1 0.2 1 02. 8

0.0 6 3 1 08. 2 1 0 3.2 Peptide-4 1 9 7.1 9 δ. 6

0.25 1 00.3 1 02. 8

0. 0 6 3 1 05. 4 1 02. 8 Peptide-5 1 1 1 7. 3 9 8. 4

0.25 1 1 8.8 1 02. 9

0.06 3 1 1 2: 8 1 02. 0 Peptide-6 1 1 1 4.δ 96.6

0.2 δ 1 1 1.8 9 2.1

0.06 3 1 05.4 99.8 Example 7

Preparation of anti-HIV peptide (peptide-1) -linked ribosome and its killing effect on HIV infected cells

Anti-I peptide (Peptide 1) 1. Dissolve 7 ^ in 1 ^ 5 OmM phosphate buffer (pH 7) and add 24/1 of 6 OmM SPDP dissolved in ethanol. After sealing, the mixture was gently stirred at room temperature for 2 hours. Next, Dithiothreitol 8.4: ^ was added to the above solution, sealed again, and gently stirred at room temperature for 2 hours. Next, this solution was applied to a Sephadex G-15 (manufactured by Pharmacia) column, and purified by gel filtration by eluting with a 5 OmM phosphate buffer (pH 7). The eluate was analyzed by reverse-phase HPLC, and the fraction eluted by gel filtration of heptide-introduced beptide 1 was collected to obtain a purified product of 1.65 ^. The conditions for the reversed-phase HPLC were as follows: Column: YMC Pack AM-303 (4.6 med. 0x25 Omm), Eluent A: 0.1% TFA, Eluent B: acetonitrile containing 0.1% TFA, Gradient: Liquid B 0% → 70% (35 minutes), flow rate: 1. O / min, detection: ultraviolet absorption at 22 Onm. On the other hand, according to the method of Martin et al. (F. J. Martin et al., Biochemistry. 20, 4229-142438, 1981), bovine brain-derived phosphatidylethanolanolamine (PE, manufactured by Sigma) 50 and SPDP Pyridyldithiopropionylphosphatidylethanolamine (PDPPE) of 31.3 and Capella 46.2 was prepared. Take 4.33 of this 0? £ into a vial, add 5 OmM phosphate buffer (pH 7.5) 1ι ^ containing the anti-HIV peptide 0.83 ^ into which the thiol group obtained above was added, and further add acetonitrile lmJ. In addition, after dissolving the entire amount, the air in the vial was replaced with nitrogen, and the peptide was bound to the phospholipids by sealing and gently stirring at 4 ° C for 16 hours. Next, the solution was transferred to a shaped flask, and the solution was evaporated by using a rotary evaporator. In addition, dimyristoylphosphatidylcholine (DMP C, Nippon Seika) 40. ng, phosphatidylglycerol from egg yolk (PG, Nippon Seika) 9.28, cholesterol (CH, Wako Pure Chemical) 9.18 vi9, And 5 m of formum methanol (9: 1) were added and dissolved and mixed. The solvent was evaporated off using a rotary evaporator to form a lipid thin film inside the flask. To this After adding 3.9 m of 1 OmM phosphate buffer (pH 7.2) containing OmM sucrose and keeping it at 30 ° C for 30 minutes to hydrate, it was stirred with a vortex mixer for 10 minutes to obtain a cloudy liquid. The obtained cloudy liquid was passed through a polycarbonate filter (manufactured by Lipex BioMembran) 10 times through a 1-inch pore size polycarbonate filter (manufactured by Nuclepore Corporation) and then 10 times through the same polycarbonate filter having a pore size of 0.2. did. This solution was subjected to gel filtration purification using a column packed with Bio-Gel A-50m (manufactured by Biorad) to obtain 26 ribosomes of the present invention to which an anti-HIV peptide (peptide-11) was bound. . The liposome obtained had a turbidity (OD ₅₄ Q) at a wavelength of 54 Onm of 1.3 and an average particle size of 1 64. Οηιη (number average particle size by light scattering method). In addition, the amount of anti-HIV peptide bound to the ribosome was quantified by reversed-phase HPLC. The conditions for reversed-phase HPLC were the same as those used for analysis of thiol-introduced anti-HIV peptides. The liposomal sample was solubilized by adding octyldarcoside to a final concentration of 50 mM, and then dithiothreitol was added to a final concentration of 5 OmM, and the mixture was kept at room temperature for 2 hours. The disulfide bond was reduced to release an anti-HIλ peptide to which a thiol group was bound. 1001 of this solution was injected into HPLC, and the total amount of the peptide in the ribosome was determined from the area of the peak of the anti-IV peptide bound to the thiol group. On the other hand, the ribosome sample was solubilized by the above method, and then injected into HPLC without reduction by dithiothreitol. Thus, the amount of the peptide encapsulated in a free form in the liposome internal aqueous layer was determined. From these analysis results, the amount of the peptide bound to the liposome was determined by the following equation. [Amount of peptide bound to ribosome] = [Total amount of peptide in ribosome solution]-[Amount of free peptide in liposomal inner aqueous layer] Anti-HIV peptide (peptide-1) bound to liposome The quantification value was 33.6 / zgZm (the number of peptide bonds per liposome was 7 Do. Next, the killing activity of the obtained ribosomes against HIV-infected cells was examined. T cell line (MOLT-4 / LAV-1) (2. δ x 10 ⁶ cells)

(RPI-1640 containing 10% fetal calf serum) to prepare a cell suspension. This cell suspension is mixed with a ribosome solution 0 having a predetermined OD ₅₄₀ by appropriately diluting with 1 OmM phosphate buffer (pH 7.2) containing 300 mM sucrose, and gently incubated at 37 ° C for 1 hour. The cells were brought into contact with the ribosome by shaking. Thereafter, a 4 m medium (RPI-1640 containing 10% fetal calf serum) was added, and the cells were cultured in a carbon dioxide incubator at 37 ° C for 3 days. After 3 said cells were stained by trypan blue staining method, and the number of viable cells was measured. Using a similar experiment using a 10 mM phosphate buffer (pH 7.2) containing 30 OmM sucrose instead of the ribosome solution as a control experiment, the growth inhibition rate was determined by the following equation. Reproduction inhibition rate (%) =

Viable cell count after 3 days of ribosome treatment

(1) x100

Viable cell count 3 days after buffer treatment (control)

A similar experiment was performed on uninfected MOLT-4 cells that were not infected with HIV. The selectivity for HIV-infected and non-infected cells was determined. The results are shown in FIG. In the figure, closed circles indicate the growth inhibition rate against HIV-infected cells (MOLT-4ZLAV-1), and open circles indicate the growth inhibition rate against uninfected cells (MOLT-4). After diluting ribosomes to OD54 ₍₎ shown in the figure, Acted on. The ribosome of the present invention showed a strong concentration-dependent growth inhibitory activity on HIV-infected cells. The liposome that inhibited the proliferation of infected cells by 50% had an OD of ₅₄ o of 0.4. On the other hand, it showed almost no growth inhibition on uninfected cells. From this, it was revealed that the ribosome of the present invention exerts a specific strong killing effect on HIV-infected cells.

Example 8

Preparation of anti-HIV peptide (peptide-2) -linked ribosome and killing effect on HIV-infected cells

In Example 7, peptide 2 was used instead of peptide 1, and thiol groups were introduced in the same manner as in Example 7 except that 1.6 was obtained. Was. The thiol group-introduced peptide—20.83 m was used as a thiol group-introduced anti-HIV peptide, and ribosomes were prepared and purified in the same manner as in Example 7 to obtain an anti-HIV peptide ( A liposome of the present invention to which peptide 2) was bound was obtained. The ribosome obtained had an OD ₅₄₀ of 1.2 and an average particle size of 157.5 nm (number average particle size by light scattering method). The amount of anti-HIV peptide (peptide-12) bound to the liposome determined by the same method as in Example 7 was 14.8 zgZm (the number of peptides bound to one ribosome was 34).

Next, the killing activity of the obtained ribosomes against HIV-infected cells was examined in the same manner as in Example 7. The results are shown in FIG. In the figure, closed circles indicate the growth inhibition rate against HIV-infected cells (MOLT-4 / LAV-1), and open circles indicate the growth inhibition rate against uninfected cells (MOLT-4). The ribosome was diluted to an OD of ₅₄ ° shown in the figure and allowed to act on each cell. The ribosome of the present invention exhibited a strong growth inhibitory activity against HIV-infected cells in a concentration-dependent manner. Of infected cells The ΔD ₅₄₀ of the ribosome that inhibits proliferation by 50% was 0.3. On the other hand, proliferation was hardly suppressed for non-infected cells.

Comparative Example 2

Preparation of anti-HIV peptide-free ribosomes and killing of HI-infected cells

In Example 7, PDPPE 4.33m9, DMP C 40.4, PG 9.28, and CH9.18 ^ were used as a ribosome membrane component without using an anti-HIV peptide, and the other methods were the same as in Example 7 to obtain ribosomes. Thus, ribosomes to which no anti-HIV peptide was bound were obtained. The liposome obtained had an OD ₅₄₀ of 1.35 and an average particle size of 169. Inm. The toxic effect of this ribosome on HIV-infected cells was examined in the same manner as in Example 7. The results are shown in FIG. In the figure, closed circles indicate the growth inhibition rate against HIV-infected cells (MOLT-4 LAV-1), and open circles indicate the growth inhibition rate against uninfected cells (MOLT-4). Incidentally, the ribosome after dilution 〇_D ₅₄₀ shown in the figure, was allowed to act on each cell. 〇_D ₅₄₀ proliferation of infected cells by 50% inhibition liposome is> 1. was 35. These results and the results obtained in Examples 7 and 8 indicate that the anti-HIV peptide of the present invention is remarkably effective in increasing the selective killing activity of ribosomes against HIV-infected cells. .

Claims

The scope of the claims

1. A peptide or a peptide derivative represented by the following sequence 1, or a peptide or a peptide derivative in which at least one of the amino acid residues of the sequence 1 is D-amino acid:

Array 1

Rj- Asp Thr Ser Glu Asp Glu Val lie Glu Lys lie Leu δ 1 0

Lys Asn Cys (R ₂ ) Lys lie Gin lie Glu Leu Asp Pro X '1 5 20

Phe Glu Asn— R ₃

twenty five

(Wherein, R, represents a hydrogen atom, a cysteine residue or a N-terminal modifying group, R ₂ represents a hydrogen atom or a cysteine modifying group, R ₃ represents a hydroxyl group, a cysteine residue or a C-terminal modifying group, X Represents a tyrosine residue or a valine residue).

2. Peptide or peptide derivative represented by the following Sequence 2 or Sequence 3 or peptide or peptide derivative in which at least one of the amino acid residues of Sequence 2 or Sequence 3 is a D-amino acid:

Array 2

R ₄ -Asp Thr Ser Glu Asp Glu Val lie Glu Lys lie Leu Lys Asn Cys (R ₂ ) Lys lie Gin lie Glu Leu Asp Pro X Phe Glu Asn- Y Array 3

Y- Asp Thr Ser Glu Asp Glu Val lie Glu Lys lie Leu Lys Asn Cys (R ₂ ) Lys lie Gin lie Glu Leu Asp Pro X Phe Glu Asn-Rs

(Wherein, R ₄ represents a hydrogen atom or a N-terminal modifying group, R ₂ represents a hydrogen atom or a cysteine modifying group, R ₅ represents a hydroxyl group or a C-terminal modifying group, and is a tyrosine residue or valine Represents a residue, where Y—Y— represents Cys—S—S—Cys— or a bifunctional crosslinking reagent).

3. The peptide or peptide derivative according to claim 1, wherein X is a tyrosine residue.

4. The peptide or peptide derivative according to claim 1, wherein X is a valine residue.

5. The peptide or peptide derivative according to claim 2, wherein X is a tyrosine residue.

6. The peptide or peptide derivative according to claim 2, wherein X is a valine residue.

7. The peptide derivative according to claim 1, represented by the following sequence 4:

Array 4

Asp Thr Ser Glu Asp Glu Val lie Glu Lys lie Leu Lys

5 10

Asn Cys (Bzl) Lys lie Gin lie Glu Leu Asp Pro Tyr Phe 15 20 2 δ G lu Asn ₀

8. The peptide derivative according to claim 1, represented by sequence 5:

Array 5

Asp Thr Ser Glu Asp Glu Val lie Glu Lys lie Leu Lys

5 10

Asn Cys (Bzl) Lys lie Gin lie Glu Leu Asp Pro Val Phe 15 20 25

Glu Asn _G

9. The peptide or peptide derivative according to claim 1, which is represented by the following sequence 6.

Array 6

Ri-D-Asp D-Thr D- Ser D-Glu D- Asp D-Glu D- Va

Five

1 D-I le D-Glu D-Lys D— lie D-Leu D- Lys D— Asn

Ten

L-Cys (R ₂ ) D-Lys D— I le D- Gin D— I le D-Glu D— Le

15 20

u D- Asp D- Pro D-Tyr D— Phe D-Glu L— Asn— R ₃

2 δ

(Wherein, represents a hydrogen atom, a cysteine residue or a N-terminal modifying group, R ₂ represents a hydrogen atom or a cysteine modifying group, and R ₃ represents a hydroxyl group, a cysteine residue or a C-terminal modifying group).

10. A pharmaceutical composition comprising the peptide or the peptide derivative according to any one of claims 1 to 9.

11. The peptide according to any one of claims 1 to 9 or The anti-HIV composition according to claim 11, which comprises a peptide derivative.

12. The composition for treating or preventing acquired immunodeficiency syndrome according to claim 11, comprising the peptide or the peptide derivative according to any one of claims 1 to 9.