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WO1994000488A1 - Peptide anti vih ou derives de ce peptide - Google Patents

Peptide anti vih ou derives de ce peptide Download PDF

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Publication number
WO1994000488A1
WO1994000488A1 PCT/JP1993/000836 JP9300836W WO9400488A1 WO 1994000488 A1 WO1994000488 A1 WO 1994000488A1 JP 9300836 W JP9300836 W JP 9300836W WO 9400488 A1 WO9400488 A1 WO 9400488A1
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WIPO (PCT)
Prior art keywords
peptide
glu
lys
lie
asp
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PCT/JP1993/000836
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English (en)
Japanese (ja)
Inventor
Kazuo Kumagai
Kazuyoshi Ikuta
Koji Ohki
Satoshi Mitsuda
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Sumitomo Pharma Co Ltd
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Sumitomo Pharmaceuticals Co Ltd
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/70514—CD4
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • 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).
  • HIV human immunodeficiency virus
  • AIDS acquired immune deficiency syndrome
  • AIDS acquired immune deficiency syndrome
  • its affinity for HIV 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. .
  • 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.
  • AZT azidothymidine
  • sulfated polysaccharide such as dextran sulfate.
  • sulfated polysaccharides are said to have problems such as low absorbability when administered orally and short blood half-life even when administered intravenously.
  • 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).
  • 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).
  • 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).
  • 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).
  • 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.
  • Lys Asn Cys (R 2 ) Lys lie Gin lie Glu Leu Asp Pro X
  • 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
  • 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
  • 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
  • X represents a tyrosine residue or palline. Represents a residue.
  • One Y—Y— represents one Cys—S—S—Cys— or a bifunctional crosslinking reagent.
  • 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.
  • 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-HI ⁇ 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.
  • 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.
  • bifunctional cross-linking reagent includes in-maleimidobenzoinole N-hydroxysuccinimido-estenole (m-maleimidobenzoyl-N-hydroxysuccinimide ester, MBS).
  • 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.
  • amino acid residues examples 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.
  • alkyl group examples 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.
  • acyl group examples 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.
  • substituent of the substituted alkyl group examples 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.
  • aromatic groups such as lower alkylamino group, di-lower alkylamino group and phenyl group, and substituted aromatic groups such as substituted phenyl group.
  • substituted aromatic substituent examples 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.
  • 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.
  • 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 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.
  • substituent of the substituted alkyl group examples 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.
  • aromatic groups such as lower alkylamino group, di-lower alkylamino group and phenyl group, and substituted aromatic groups such as substituted phenyl group.
  • substituted aromatic substituent examples 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 peptide synthesis method by a solid phase method is suitable.
  • a method using Fmoc amino acid E. Atherton and ⁇ ⁇ 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.
  • Fmoc 9-fluorenylmethyloxycarbonyl
  • the Fmoc amino acid residue is sequentially bonded by a condensation reaction.
  • 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.
  • 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. .
  • 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.
  • HPLC high performance liquid chromatography
  • 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.
  • 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.
  • 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).
  • 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.
  • thiol groups derived from cysteine residues are present in the peptide
  • the thiol group in the peptide can be used as it is.
  • 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.
  • 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.).
  • 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.
  • 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.
  • the formed liposome can hold a drug in its inner aqueous layer, and is therefore useful as a drug delivery body.
  • a toxin for example, diphtheria toxin fragment A
  • an anti-HIV agent such as AZT
  • 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.
  • the cytotoxic substance can be selectively transmitted to HIV-infected cells. And can selectively kill the infected cells.
  • proteinaceous cytotoxic substances that inhibit cellular protein synthesis include ricin, gelonin, diphtheria toxin, Pseudomonas aeruginosa exotoxin A, and toxicologically active fragments thereof.
  • a thiol group is introduced into both the peptide and the cytotoxic substance using the aforementioned bifunctional crosslinking reagent such as SPDP.
  • the aforementioned bifunctional crosslinking reagent such as SPDP.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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 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 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.
  • 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 1 Fmoc-D-Val as a Fmoc amino acid residues, Fmoc-D-l ie ⁇ 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.
  • 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 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
  • 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 TM resin.
  • the other steps were the same as in Example 1 to synthesize, purify, and analyze the amino acid sequence.
  • 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.
  • 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.
  • 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.
  • HIV HTLV-mB
  • various concentrations (2x l 0 3 TC ID 5 .)
  • 4 days at 37 ° C for with Ml 0 cells are CD 4-positive cells (10 4 cells)
  • 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.
  • 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.
  • 401 medium RPM I-1640 medium supplemented with 10% fetal bovine serum
  • 101 thiazolyl blue tetrazolium umb. MTT thiazolyl blue tetrazolium umb.
  • 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.
  • Martin et al. F. J. Martin et al., Biochemistry.
  • PE bovine brain-derived phosphatidylethanolanolamine
  • SPDP Pyridyldithiopropionylphosphatidylethanolamine (PDPPE) 31.3 and Capella 46.2 was prepared.
  • PDPPE Pyridyldithiopropionylphosphatidylethanolamine
  • the solvent was evaporated off using a rotary evaporator to form a lipid thin film inside the flask.
  • a rotary evaporator 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. ⁇ (number average particle size by light scattering method).
  • 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-HI ⁇ 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.
  • T cell line (MOLT-4 / LAV-1) (2. ⁇ 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.
  • 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.
  • 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).
  • Example 7 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 ⁇ D 540 of the ribosome that inhibits proliferation by 50% was 0.3. On the other hand, proliferation was hardly suppressed for non-infected cells.
  • 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.
  • 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.

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Abstract

Peptide, ou dérivé de ce peptide, représenté par la séquence (1), ou peptide ou dérivé de ce peptide dans lequel au moins un acide aminé de la séquence (1) est un acide aminé D, et un dimère de cette substance: 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, dans laquelle R1 représente l'hydrogène, un résidu de cystéine ou un groupe modificateur à terminaison N; R2 représente l'hydrogène ou un groupe modificateur cystéine; R3 représente hydroxy, un résidu de cystéine ou un groupe modificateur à terminaison C; et X représente un résidu de tyrosine ou de valine. Ce peptide ou ce dérivé de peptide a un effet inhibiteur sur l'infection par le virus de l'immunodéficience humaine (VIH) et sur la formation de cellules géantes multinucléées après l'infection par VIH, ce qui le rend utile pour le traitement et la prévention du syndrome d'immunodéficience acquise.
PCT/JP1993/000836 1992-06-23 1993-06-22 Peptide anti vih ou derives de ce peptide Ceased WO1994000488A1 (fr)

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JP4/164808 1992-06-23
JP16480892 1992-06-23

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Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5603933A (en) * 1993-08-31 1997-02-18 Board Of Regents, The University Of Texas CD4 peptides for binding to viral envelope proteins
US5852007A (en) * 1995-11-28 1998-12-22 Cephalon, Inc. Cysteine and serine protease inhibitors containing D-amino acid at the P2 position, methods of making same, and methods of using same
US6210873B1 (en) 1987-08-28 2001-04-03 Board Of Regents, The University Of Texas System Methods and compositions for the priming of specific cytotoxic T-lymphocyte response
US6265539B1 (en) 1987-08-28 2001-07-24 The University Of Texas System The Board Of Regents Prophylaxis and therapy of acquired immunodeficiency syndrome
AU2003200161B2 (en) * 1997-07-24 2004-12-02 Curis, Inc. Method of treating dopaminergic and gaba-nergic disorders
JP2023514447A (ja) * 2020-02-21 2023-04-05 アシスタンス パブリック－ホピトー デ パリ ペプチド－メラニン結合の最適化

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JPH02131497A (ja) * 1988-05-27 1990-05-21 Ortho Pharmaceut Corp Ｈｉｖ―１のｃｄ４受容体蛋白質への結合をブロツクするペプチド
JPH02152989A (ja) * 1988-12-02 1990-06-12 Calpis Food Ind Co Ltd:The 抗ｈｉｖペプチド
JPH0338599A (ja) * 1989-07-05 1991-02-19 Calpis Food Ind Co Ltd:The 抗ｈｉｖペプチド及び抗ｈｉｖ修飾ペプチド

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JPH02131497A (ja) * 1988-05-27 1990-05-21 Ortho Pharmaceut Corp Ｈｉｖ―１のｃｄ４受容体蛋白質への結合をブロツクするペプチド
JPH02152989A (ja) * 1988-12-02 1990-06-12 Calpis Food Ind Co Ltd:The 抗ｈｉｖペプチド
JPH0338599A (ja) * 1989-07-05 1991-02-19 Calpis Food Ind Co Ltd:The 抗ｈｉｖペプチド及び抗ｈｉｖ修飾ペプチド

Non-Patent Citations (1)

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Title
CELL, Vol. 42, (1985), P.J. MADDON, "The Isolation and Nucleotide Sequence of a cDNA Encoding the T-Cell Surface Protein T4: A New Member of the Immunoglobulin Gene Family", pp. 93-104. *

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6210873B1 (en) 1987-08-28 2001-04-03 Board Of Regents, The University Of Texas System Methods and compositions for the priming of specific cytotoxic T-lymphocyte response
US6265539B1 (en) 1987-08-28 2001-07-24 The University Of Texas System The Board Of Regents Prophylaxis and therapy of acquired immunodeficiency syndrome
US5603933A (en) * 1993-08-31 1997-02-18 Board Of Regents, The University Of Texas CD4 peptides for binding to viral envelope proteins
US5852007A (en) * 1995-11-28 1998-12-22 Cephalon, Inc. Cysteine and serine protease inhibitors containing D-amino acid at the P2 position, methods of making same, and methods of using same
AU2003200161B2 (en) * 1997-07-24 2004-12-02 Curis, Inc. Method of treating dopaminergic and gaba-nergic disorders
JP2023514447A (ja) * 2020-02-21 2023-04-05 アシスタンス パブリック－ホピトー デ パリ ペプチド－メラニン結合の最適化

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