WO2014178680A1 - Peptide d'inhibition de l'expression hsp et composition le contenant - Google Patents

Peptide d'inhibition de l'expression hsp et composition le contenant Download PDF

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WO2014178680A1
WO2014178680A1 PCT/KR2014/003948 KR2014003948W WO2014178680A1 WO 2014178680 A1 WO2014178680 A1 WO 2014178680A1 KR 2014003948 W KR2014003948 W KR 2014003948W WO 2014178680 A1 WO2014178680 A1 WO 2014178680A1
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composition
hsp
peptide
pep
inhibiting
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PCT/KR2014/003948
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김상재
김범준
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주식회사 카엘젬백스
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Priority to KR1020157031591A priority Critical patent/KR102275912B1/ko
Publication of WO2014178680A1 publication Critical patent/WO2014178680A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents

Definitions

  • HSP expression inhibitory peptide and composition comprising same
  • the present invention relates to a peptide having HSP expression inhibitory activity and a composition comprising the same, and more particularly to a peptide having HSP expression inhibitory activity as a peptide derived from telomerase and a composition comprising the same, and It relates to a method of inhibiting HSP expression.
  • Cancer cells express high levels of several Heat Shock Proteins (HSPs) to enhance tumor aggression and allow cells to survive mortality conditions, such as by treatment.
  • HSPs Heat Shock Proteins
  • elevated HSP expression inhibits programmed cell death and promotes autologous growth resulting in cancer cell growth.
  • thermal stratified protein has been known to be a cell constituent protein that acts as a molecular chaperone and protects cells in stress environments such as heat, hypoxia and radioactivity (Ritossa F. Experimentia 1962; 18: 571-3; Lindquist S et al., Craig Annu Rev Genet 988; 22: 631-77). Recently cell growth and
  • HSP heat shock proteins
  • HSP 70 has increased expression in endometrial cancer, malignant osteosarcoma, and kidney cancer, which is thought to be due to the inhibition of apoptosis.
  • HSP 90 accounts for 1- of normal cellular proteins and increased expression, particularly in stress environments. HSP 90 reacts with ATP to fold denatured proteins and has increased expression in lung cancer, leukemia, Hodgkin's disease, etc., and HSP 90 has also been reported to be associated with apoptosis, but the exact role has not been identified. not.
  • HSP70 is a highly conserved protein chaperone associated with many intracellular mechanisms.
  • HSP70 is induced by intracellular stress and suppresses stress-induced apoptosis
  • HSP70 has immunomodulatory capacity and is known to stimulate the production of anti-inflammatory cytokines (Van Eden, W. et al., Nat. Rev. Immunol.
  • TNF tumor necrosis factor
  • APCs Antigen presenting cells
  • HSP70 (constitutively expressed Hsc70 and heat induced HSP70-1 and HSP70-6) is involved in the preliminary stage, while HSP90 is involved in the last stage.
  • Their functions include a number of co-chaperones such as HSP70-regulators, HSP40, HSP110, BAG and HIP; Requires HSP-organizing protein (HOP), which is involved in the formation of intermediate molecular chaperone complexes, where the client passes from HSP70 to HSP90, and other such as p23, cdc37 and immunophilin in the final or mature HSP90 complex Works.
  • HSP90 is recognized as a new anticancer target.
  • HSP90 is a highly ubiquitous (1-2% of whole cell protein) essential protein that functions as a molecular chaperone to ensure morphological stability, shape and function of the client protein. Inhibition of its native ATPase activity of HSP90 disrupts HSP90-client protein interactions, leading to its degradation via the ubiquitin proteasome pathway.
  • Subtypes of the HSP90 client protein such as Raf, AKT, CDK4, and ErbB2 are EGFR family of oncogenic signaling molecules that are critically involved in cell growth, differentiation and apoptosis. It is important in Pau
  • the present inventors have completed the present invention as a result of intensive efforts to develop a drug capable of specifically binding to HSP and inhibiting the expression of HSP.
  • the inventors have developed a peptide that specifically binds to HSP as a peptide derived from telomerase and can specifically inhibit the expression of HSP.
  • An object of the present invention is to provide a peptide that binds to HSP and inhibits the expression of HSP and a method for inhibiting HSP expression using the same.
  • composition for inhibiting HSP expression comprising a peptide comprising the amino acid sequence of SEQ ID NO: 1, a peptide having a sequence homology of 80% or more with the amino acid sequence or a fragment thereof.
  • the HSP may be HSP 70 or HSP 90.
  • the fragment may be a fragment consisting of three or more amino acids.
  • the peptide may be composed of up to 30 amino acids.
  • the composition is tumor growth and metastasis, diabetic retinopathy, prematurity retinopathy, corneal graft rejection, neovascular glaucoma, melanoma proliferative retinopathy, psoriasis, macular degeneration ( macular degeneration, hemophilia Capillary proliferation in sexual joints, atherosclerotic plaques, keloids, wound granulation, vascular adhesion, rheumatoid arthritis, chronic inflammation, osteoarthritis, autoimmune disease, Crohn's disease, restenosis, atherosclerosis Hardening, intestinal adhesion, cat scratch disease, ulcers, cirrhosis, glomerulonephritis, diabetic nephropathy, malignant neurosis, thrombotic microangiopathy, organ transplant rejection, renal glomerulopathy, diabetes, inflammatory or neurodegenerative diseases Sex angiogenesis-may be used for the prevention or treatment of
  • the composition for inhibiting angiogenesis may be used for the prevention or treatment of tumor growth and metastasis.
  • the composition may be a pharmaceutical composition.
  • the composition may be a food composition.
  • it may be a method for preventing and treating HSP expression related diseases, including a system for administering the above-mentioned composition to a subject.
  • a method capable of effectively inhibiting tumor growth may be provided by using a template that specifically binds to HSP.
  • PEP 1 is to identify a protein that interacts with PEP 1 as follows;
  • each protein complex was purified using Ni-NTA chromatography and then subjected to SDS-PAGE and silver staining. Proteins specifically associated with PEP 1-GFP were confirmed by Maldi-TOF mass spectrometry after excision on gels. * Means GFP, llmer-GFP, PEP 1-GFP bands.
  • Identified proteins associated with PEP 1 may be immunized with antibodies.
  • Lysates of HepG2 cells were incubated with GST or PEP 1-GST proteins.
  • the results of the pull down using glutathione resin and the results of HSP70 and PEP 1 were confirmed using immunoblotting and anti-HSP70 antibody.
  • FIG. 2 to 3 show the down regulation of HSP70 and HSP90 according to PEP 1 treatment in cancer cells.
  • Jurkat FIG. 2
  • MCF7 FIG. 3
  • the amount of protein of HSP70 and HSP90 was analyzed by immunoblotting using antibodies to HSP70, HSP90 and GAPDH.
  • MCF7 and HeLa cells were treated with PEP 1 (20 ⁇ ) or a solvent and incubated at low oxygen for a specified time. Cell lysates were subjected to immunoblotting to analyze the amounts of HSP70 and HSP90.
  • FIG. 6 shows the results of treating solvent, PEP 1, 17-AAG, and KNK437 on Jurkat and MCF7 cells.
  • Jurkat and MCF7 cells were treated with vehicle, PEP 1 (5 ⁇ for Jurkat and 20 ⁇ for MCF7), 17-AAG (1 ⁇ ), NK437 (1 ⁇ ) in serum-free medium for 2 hours.
  • Cell lysates were analyzed by immunoblotting similar to the method used in FIG. 2.
  • FIG. 7 shows the results of treatment of MCF7 cells with PEP 1 or PBS in both MG132 (5 ⁇ ) and non-containing states.
  • Intracellular HSPs and cell surface HSPs were stained by surface intracellular staining and surface staining staining, and analyzed using flow cytometry as described in the test materials and methods.
  • Red means DMS0, Blue: DMS0CPEP 1 plus DMS0 with PEP 1; Orange: MG132 with PEP 1 (PEP 1 plus MG132); Green: Means MG132 do.
  • FIGS. 8 to 9 are graphs showing inhibition of cancer cell proliferation by PEP 1 in a low oxygen state.
  • MCF7 and HeLa cells were cultured with or without PEP 1 in either normal oxygen (left panel) or low oxygen (right panel). At 2, 4 and 6 days of culture, the number of cells was measured. Data are mean ⁇ standard deviation (Data represent means ⁇ SD). *: p ⁇ 0.05 was considered statistically significant. A one-way t-test was performed.
  • HSP70 and HSP90 protein levels of tumors by PEP 1 treatment show the reduction of HSP70 and HSP90 protein levels of tumors by PEP 1 treatment.
  • HSP70 and HSP90 protein levels in tumor sections were visualized by immunohistochemical staining with antibodies against HSP70 and HSP90 (FIG. 10) and quantified using Leica Qwin software (FIG. 10).
  • FIG. 14 the correlation between HSP70 level in blood and tumor weight (left panel) or tumor size (right panel) was analyzed. A binary t-test was performed.
  • the present invention may be variously modified and have various embodiments.
  • the present invention will be described in more detail with reference to examples.
  • these examples are not intended to limit the present invention to a particular embodiment, the present invention is capable of various examples and uses on the basis of the claims are included in the spirit and scope of the present invention It should be understood to include all transformations, equivalents, or substitutes that are made.
  • the detailed description of the related known technology may obscure the gist of the present invention, the detailed description thereof will be omitted.
  • Telomere is a genetic material repeatedly present at the end of a chromosome and is known to prevent damage to the chromosome or binding to another chromosome. Each time a cell divides, the telomeres get a little shorter. In the presence of fever, telomeres become very short, and the cells stop dividing and die. On the other hand, elongation of telomeres is known to prolong cell life. For example, cancer cells secrete an enzyme called telomerase, which prevents telomeres from shortening . Be known
  • HSP90 family protein As the compound that binds to the HSP90 family protein, benzoquinone anamycin-based antibiotics such as geldanamycin and habimycin, and radicicol are known. All these compounds are reported to exhibit pharmacological activity such as antitumor activity by binding to HSP90 family protein and inhibiting the function of HSP90 family protein. Therefore, compounds that bind to HSP90 family proteins are known to be useful as therapeutic agents for diseases involving HSP90 family proteins or proteins to which HSP90 family proteins bind (HSP90 client proteins).
  • One aspect of the present invention provides a peptide comprising a amino acid sequence of SEQ ID NO: 1, a polynucleotide encoding a peptide that is a peptide or fragment thereof having a sequence homology of 80% or more with the amino acid sequence.
  • the polynucleotides can be used to mass produce peptides. For example, a large amount of peptides can be produced by culturing a vector containing a polynucleotide encoding a peptide into a host cell.
  • the peptides disclosed herein may include peptides having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% homology. have.
  • the peptide disclosed herein the peptide comprising SEQ ID NO: 1 or fragments thereof and one or more amino acids, two or more amino acids, three or more amino acids, four or more amino acids, five or more amino acids, six or more amino acids Or peptides with seven or more amino acids changed.
  • amino acid changes belong to the property that allows the physicochemical properties of the peptide to be altered.
  • amino acid changes can be made, such as improving the thermal stability of the peptide, altering substrate specificity, changing the optimal pH, and the like.
  • a peptide comprising the sequence of SEQ ID NO: 1 or a peptide having at least 80% sequence homology with the peptide sequence is selected from telomerase, specifically ⁇ Homo sapiens telomerase. Peptides derived.
  • amino acid refers to 22 amino acids that are naturally incorporated into a peptide. Standard amino acids as well as D-isomers and modified amino acids. Accordingly, in one aspect of the invention the peptide may be a peptide comprising D-amino acids. On the other hand, in another aspect of the invention, the peptide may include post-trans 1 at ional modification, non-standard amino acid and the like . have. Of posttranslational modifications. Examples include phosphorylation, glycosylation, acyllat ion (eg, acetylation, myristoylated yr istoylat ion) and
  • Palmitoylation alkylat ions
  • chemical properties e.g., beta- Removal deimidization, deamidation
  • structural changes e.g, formation of disulfide bridges
  • Changes in amino acids such as changes in amino acids, such as amino groups, carboxyl groups or side chains, caused by chemical reactions occurring in the process of bonding with crosslinkers.
  • Peptides disclosed herein can be wild-type peptides identified and isolated from a natural source.
  • the peptides disclosed herein may be artificial variants comprising amino acid sequences in which one or more amino acids have been substituted, deleted and / or inserted compared to peptides that are fragments of SEQ ID NO: 1.
  • Amino acid changes in wild type polypeptides as well as in artificial variants include conservative amino acid substitutions that have no significant effect on the folding and / or activity of the protein.
  • conservative substitutions include basic amino acids (arginine, lysine and histidine), acidic amino acids (glutamic acid and aspartic acid), polar amino acids (glutamine and asparagine), hydrophobic amino acids (leucine, isoleucine, valine and methionine), aromatic amino acids ( Phenylalanine, tryptophan and tyrosine), and small amino acids (glycine, alanine, serine and threonine). Amino acid substitutions that generally do not alter specific activity are known in the art.
  • hydrophobicity norleucine, met, ala, val, leu, ile
  • Non-conservative substitutions may be made by exchanging a member of one of these classes for another class. Will be done. Any cysteine residue that is not involved in maintaining the proper conformation of the peptide can generally be substituted with serine to improve the oxidative stability of the molecule and to prevent abnormal crosslinking. Conversely, cysteine bond (s) can be added to the peptide to improve its stability.
  • Another type of amino acid variant of the peptide is that the glycosylation pattern of the antibody is altered.
  • ⁇ will.
  • change is meant deletion of one or more carbohydrate residues found in the peptide and / or addition of one or more glycosylation sites that are not present in the peptide.
  • N-linked refers to a carbohydrate moiety attached to the side chain of an asparagine moiety.
  • the tripeptide sequences asparagine -X-serine and asparagine -X-threonine, where X is any amino acid except proline, are recognition sequences for enzymatic attachment of carbohydrate moieties to the asparagine side chains.
  • X is any amino acid except proline
  • 0-linked glycosylation means attaching one of the sugars N-acetylgalactosamine, galactose or xylose to hydroxyamino acids, most commonly serine or threonine, but the 5-hydroxyproline is lean or 5-hydroxy Roxylysine can also be used.
  • glycosylation sites to the peptide is conveniently carried out by changing the amino acid sequence containing one or more of the above-mentioned tripeptide sequences (for N-linked glycosylation sites). Such changes may be made by adding one or more serine or threonine residues to the sequence of the original antibody or by substituting these residues (for a 0-linked glycosylation site).
  • the polynucleotide may be a naturally occurring or artificial DNA or RNA molecule as a nucleic acid molecule, and may be single stranded or double stranded.
  • the nucleic acid molecule may be one or more, which may be nucleic acid molecules of the same type (eg, having the same nucleotide sequence), or may be nucleic acid molecules of other types.
  • a peptide comprising the amino acid sequence of SEQ ID NO: 1, a peptide having 80% or more sequence homology with the amino acid sequence, or a fragment thereof is administered to a subject in need of inhibition of HSP expression.
  • HSP expression inhibition method comprising the.
  • Another aspect of the present invention provides a peptide comprising an amino acid sequence of SEQ ID NO: 1 in preparing a composition for inhibiting HSP expression, a peptide having a sequence homology of 80% or more with the amino acid sequence, or a fragment thereof Phosphorus peptides.
  • SEQ ID NO: 1 (hereinafter 'PEP 1') in the present invention is a telomerase-derived peptide, a peptide consisting of 16 amino acids as described below.
  • the peptides set forth in SEQ ID NO: 1 are shown in Table 2 below. "Name” in Table 2 below is named to distinguish the peptide.
  • the peptide set forth in SEQ ID NO: 1 represents the entire peptide of human telomerase.
  • a peptide having a sequence of SEQ ID NO: 1, a peptide that is a fragment of the sequence of SEQ ID NO: 1, or a peptide having a sequence homology of 80% or more with the peptide sequence is a peptide contained in telomerase. It includes the "synthetic peptide" synthesized by selecting the peptide of the corresponding position.
  • SEQ ID NO: 2 shows the amino acid sequence of the entire telomerase.
  • An aspect of the present invention provides a method for inhibiting HSP expression for delivering at least one active ingredient of a peptide comprising SEQ ID NO: 1 or fragments thereof or a peptide having 80% or more sequence homology with the peptide sequence. It provides a composition for.
  • ⁇ 84> in one aspect of the invention comprises a peptide or fragment thereof which are peptides de, or conjugates of HSP binding peptides as the active ingredient having the peptide sequence with at least 80% sequence homology comprising SEQ ID NO: 1,
  • a pharmaceutical or food composition having an excellent effect of delivering an active ingredient into cells.
  • composition according to one aspect of the present invention comprises a peptide comprising an amino acid sequence of SEQ ID NO: 1 in one aspect, a peptide having a sequence homology of 80% or more with the amino acid sequence, or a fragment thereof.
  • jg / mg to 1 mg / mg specifically 1 Ug / nig to 0.5 mg / mg, more specifically 10 // g / mg to 0.1 mg / mg.
  • composition according to one aspect of the present invention may be applied to all animals including humans, dogs, chickens, pigs, cattle, sheep, guinea pigs or monkeys.
  • the pharmaceutical composition according to the uniaxial aspect of the present invention may be administered orally, rectally, transdermally, intravenously, intramuscularly, intraperitoneally, intramedullary, intradural or subcutaneously.
  • ⁇ 88> 'formulation for oral administration is not the form of tablets, pills, soft or hard shell selje, granules, powders, solutions or emulsions, but can best, limited.
  • Formulations for parenteral administration may be, but are not limited to, injections, drops, lotions, ointments, gels, creams, suspensions, emulsions, suppositories, patches or sprays.
  • the pharmaceutical composition according to one aspect of the present invention may include additives of diluents, excipients, lubricants, binders, disintegrants, laxatives, dispersants, surfactants, colorants, fragrances or sweeteners as needed.
  • Pharmaceutical compositions according to one aspect of the invention may be prepared by conventional methods in the art.
  • the pharmaceutical composition according to one aspect of the present invention may be administered once to three times a day, but is not limited thereto.
  • the formulation of the food composition according to one aspect of the present invention is not particularly limited, but may be, for example, formulated into a tablet, a granule, a powder, a liquid, a solid formulation, and the like.
  • Each formulation can be appropriately selected and blended by those skilled in the art according to the formulation or purpose of use, in addition to the active ingredient, and synergistic effects may occur when applied simultaneously with other raw materials.
  • the daily dosage of the active ingredient is within the level of one skilled in the art, the daily dosage of which is for example specifically 1 / zg / kg / day to 10 mg / kg / day, more specifically 10 / kg / day to 1 mg / kg / day, and more specifically 50 / kg / day to 100 // g / kg / day, but is not limited thereto, and includes the age, health condition, It can depend on a variety of factors, including complications.
  • Preferred embodiments of the invention include the most optimal mode known to the inventors for carrying out the invention. Variations of the preferred embodiments may become apparent to those skilled in the art upon reading the foregoing description. We believe that those skilled in the art will appreciate such variations. In anticipation of use, the inventors expect the invention to be practiced in a manner different from that described herein. Accordingly, the invention includes all modifications and equivalents of the subject matter referred to in the appended claims, as permitted by patent law. Moreover, any and all combinations of the above-mentioned components within all possible variations are included in the present invention unless expressly stated to the contrary or apparently contradictory in context. While the invention has been particularly shown and described with reference to exemplary embodiments, those skilled in the art will appreciate that various changes in form and detail may be made without departing from the spirit and scope of the invention as defined by the following claims. .
  • PEP 1 '' The peptide of SEQ ID NO: 1 (hereinafter referred to as "PEP 1 '') was prepared according to the conventionally known solid phase peptide synthesis method. Specifically, the peptides were prepared using Fmoc using ASP48S (Peptron, Inc., Daejeon, Korea). It was synthesized by coupling one amino acid from the C-terminus to one through solid phase peptide synthesis (SPPS), using the first amino acid at the C-terminus of the peptides attached to the resin, for example:
  • PEP 1 prepared in the same manner as in Example 1 was used to experiment with binding to HSP and HSP inhibition.
  • Human breast cancer cell lines MCF7 Human breast adenocarcinoma cell line
  • human T lymphocyte cell line human T lymphocyte cell line (Jurkat)
  • MC38 murine colon adenocarcinoma
  • HeLa human cervical adenocarcinoma
  • HeLa human cervical adenocarcinoma
  • DMEM Dulbecco's modified Eagle's medium
  • MCF7 and HeLa cells were incubated with low oxygen and normal oxygen after treatment with 20 ⁇ PEP 1.
  • Anoxia was induced using BBL GasPak (Becton Dickinson), which produced a catalyst reaction within 90 minutes to reduce oxygen to undetectable levels. Incubation time ranged from 2 to 24 hours.
  • cells were harvested and subjected to immunoblotting using ci-HSP70, Q-HSP90, ⁇ -HIF-la, or a -GAPDH antibodies.
  • a -GAPDH was used to normalize the amount of HSP70 / 90 to the amount of GAPDH for protein quant i f icat ion.
  • MCF7 and HeLa cells were inoculated in a 96 well plate to be ⁇ cells per well, and then cultured in 37 ° C. and 5% C0 2 conditions in complete media with 10% FBS. After serum starvation treatment for 2 hours, the cells were cultured in all of the complete medium including PEP 1 (20 ⁇ ). As described above, the cells were low acid for 1 to 6 days. Cultures were carried out in small or normal oxygen. The number of viable cells was measured daily using trypan blue staining method. All calculation experiments were performed in duplicate.
  • Jurkat and MCF7 cells (5 ⁇ 10 5 ) were inoculated and cultured for 12 hours.
  • the samples were a-HSP70 (sc-32239 and sc-66048, Santa Cruz, CA, USA), a -HSP90 (abl429, abeam, USA), ⁇ -GRP78 (sc-13968, Santa Cruz, CA, USA), ⁇ -HIF-la (sc-10790, Santa Cruz, CA, USA) or a -GAPDH (sc-25778, Santa Cruz, CA, USA) antibodies were used to perform SDS-PAGE and immunoblotting. Immune semi-male bands were visualized using an enhanced chemiluminescence kit (cha luminescence kit, iNtRoN Biotechnology, INC, Korea) and quantified using ImageQuantTM LAS-4000 (GE Healthcare Life Science, NJ, US).
  • an enhanced chemiluminescence kit cha luminescence kit, iNtRoN Biotechnology, INC, Korea
  • proteasome inhibition test cells were treated with 5 ⁇ M of proteasome inhibitor MG132 (Calboicam) during the culture. Cells were separated using trypsin and washed with cold PBS (phosphate buffered saline) and FACS buffer (PBS containing 1% BSA and 0.1% NaN 3 ). Cells were treated with permeabilization buffer (eBioscience, CA, USA) according to the manufacturer's instructions for intracellular staining. Cells were reacted with a-HSP70-FTC (ab61907, Abeam) or a-HSP90-PE (ab65171, Abeam) at 4 ° C for 30 minutes. Flow cytometry was performed using flow cytometry FACScan (FACScan flow cytometer, Bee ton Dickinson Co., CA, USA). Data was analyzed using Flovjo TM software (version 10.0.5, Tree Star, Inc., OR, USA).
  • mice 7 week old BALB / c athymic (athymic) (Nu / Nu) mice (10 mice per group;
  • HSP70 and HSP90 proteins in tumors were evaluated using immunohistochemical staining (i ⁇ unohistochemkal staining) in a similar manner to PCNA staining.
  • Antibodies against heat stratified protein (HSP70; sc-7298, HSP90; abl429) were used as primary antibodies All. Expression of HSP70 and HSP90 proteins by tumors was assessed by immunoblotting using tumor lysate.
  • the tumors were ground using a mortar and pestle, followed by extract ion buffer, 20 mM HEPES, pH7.5, 100 mM NaCl, 0.05% Triton X-100, ImM DTT, 0.5 mM sodium orthovanadate, 1 mM EDTA, 0.5 mM PMSF, 10 ⁇ g / ml aprot inin, 5 ⁇ g / ml leupept in, 2 yg / ml pepstatin). After repeated centrifugation, the supernatant was subjected to SDS-PAGE and immunoblotting (i ⁇ unoblotting) as described above.
  • extract ion buffer 20 mM HEPES, pH7.5, 100 mM NaCl, 0.05% Triton X-100, ImM DTT, 0.5 mM sodium orthovanadate, 1 mM EDTA, 0.5 mM PMSF, 10 ⁇ g / ml aprot inin, 5 ⁇
  • VEGF secretion of cancer cells was confirmed by ELISA (Enzyme-1 inked i ⁇ unosorbent assay). MCP7 and HeLa cells were cultured in low or normal oxygen state after adding PEP 1 or vehicle for 24 hours. The amount of VEGF in the cell supernatant was determined using the human VEGF immunoassay kit (R & D Systems, USA) according to the manufacturer's instructions. To analyze the concentrations of HSP70 and HSP90 in the blood, blood was collected from a mouse model with tumors. After serum preparation, the concentrations of HSP70 and HSP90 in the blood were confirmed using immunoassay kits of HSP70 (R & D systems, USA) and HSP90 (Cusabio Biotech co., Ltd, DE, USA.).
  • the sliced tumor sections were fixed for 15 minutes at 4 ° C. with paraformaldehyde at room temperature. After washing twice with PBS, it was placed in PBS containing 0.25% Triton X-100 for 10 minutes, and then washed three times again with PBS. After 30 min blocking of tissue with 1% BSA-PBST, 4 ° C wet with a combination of mouse anti-Tie2 (557039, BD Pharmigen) and rat anti-CDllb antibodies (ab8878, abeam) Incubated in chamber. After washing, the tissues were incubated with a mixture of AlexaFlour 488 goat anti-mouse IgG and AelxaF lour 633 goat anti-rat IgG. In order to visualize the cell nuclei, the cells were treated with DAPI (Sigma Aldrich) for 1 minute and analyzed by confocal microscopy.
  • DAPI Sigma Aldrich
  • Example 3 Screening of Binding Proteins of PEP 1 via Pull Down Assay The pool pull experiment to identify proteins interacting with PEP 1 comprises PEl—GFP. Was carried out using a His-tag. PEP 1-GFP was incubated with total cell lysates of HEK293T cells (Korea Cell Line Bank), followed by Ni-NTA affinity chromatography.
  • GFP protein and PEP 1 peptide fused with GFP were used as a control.
  • Eluted protein was isolated by 12% SDS-PAGE and observed by silver staining (catalog # PR0T-SIL1, Sigma Aldrich, MO, USA). Protein bands showing specific interactions were excised from the gels and analyzed by MALDI-TOF (Proteomix, Seo, Korea) for their identification.
  • GFP-PEP 1 or GFP protein is incubated with whole cell lysate of HEK293T cells. Each protein complex is purified and subjected to SDS-PAGE and silver staining experiments. Several proteins were specifically found in the PEP 1-GFP protein complex. However, it was not found in the GFP protein or GFP-PEP 1 complex.
  • PEP 1-GST was able to successfully pull down HSP70 from cell lysates of MCF7 cells and HepG2 cells (FIG. 1C). Subsequently, fragments of MCF7 cell membranes were purified and incubated with GFP or GFP-PEP 1. As can be seen in Figure 1D, HSP70 was associated with GFP-PEP 1 and precipitated with GFP-PEP 1. This results in the HSP70
  • HSP90 decreased by up to 20% in the PEP 1-treated group at 5 ⁇ compared to the control.
  • HSP70 decreased by about 50% compared to the control group.
  • treatment with scrambled peptides similar to PEP 1 but with different sequences did not significantly affect the levels of HSP70 and HSP90 (see FIGS. 2 and 3).
  • 17-AAG and NK437 reduced the amount of HSP90 but not HSP70.
  • PEP 1 and KNK437 reduced the amount of both HSP90 and HSP70, while 17-MG had little effect on the levels of HSP90 and HSP70.
  • PEP 1 The effect of PEP 1 was investigated. PEP 1 showed a weak inhibitory effect on MCF7 and HeLa cell growth under normal conditions (Normoxia, normal oxygen condition), but the inhibitory effect of PEP 1 was greatly enhanced in hypoxic conditions (see FIGS. 8 and 9).
  • HSP70 and HSP90 can be secreted from tumor cells, and recent studies have shown some role in tumorigenicity and antitumor reaction.
  • HSP70 and The concentration of HSP90 was measured. As a result of the measurement, it was observed that the PEP 1 treatment group had a great effect on the concentration decrease ⁇ of HSP70. (See Figure 13),

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Abstract

La présente invention fournit une composition d'inhibition d'une expression HSP, contenant un peptide comprenant une séquence d'acides aminés de SEQ ID no : 1, un peptide ayant une similitude de séquence qui est supérieure ou égale à 80 % de la séquence peptidique, ou un peptide de liaison à l'HSP, qui est un fragment de celle-ci, et un procédé d'inhibition d'une expression HSP l'utilisant. Particulièrement, en raison qu'une HSP augmente spécifiquement dans une cellule cancéreuse, l'invention permet d'obtenir un procédé d'inhibition d'une expression HSP en utilisant un peptide se liant spécifiquement à l'HSP.
PCT/KR2014/003948 2013-05-03 2014-05-02 Peptide d'inhibition de l'expression hsp et composition le contenant WO2014178680A1 (fr)

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EP3011967A4 (fr) * 2013-06-21 2017-03-29 Gemvax & Kael Co., Ltd. Régulateur de sécrétion d'hormone, composition le contenant, et procédé pour contrôler une sécrétion d'hormone l'utilisant

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