WO2023033128A1 - Method for producing protein - Google Patents
Method for producing protein Download PDFInfo
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- WO2023033128A1 WO2023033128A1 PCT/JP2022/033054 JP2022033054W WO2023033128A1 WO 2023033128 A1 WO2023033128 A1 WO 2023033128A1 JP 2022033054 W JP2022033054 W JP 2022033054W WO 2023033128 A1 WO2023033128 A1 WO 2023033128A1
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- WIPO (PCT)
- Prior art keywords
- pkc
- cells
- nucleic acid
- culturing
- alkyl
- Prior art date
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Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P21/00—Preparation of peptides or proteins
- C12P21/02—Preparation of peptides or proteins having a known sequence of two or more amino acids, e.g. glutathione
Definitions
- biopharmaceuticals have made it possible to treat many chronic and acute diseases that were previously difficult to treat, and are becoming essential pharmaceuticals for modern medicine.
- the price of biopharmaceuticals is very high, and many people cannot enjoy the benefits.
- the biggest reason for this is that cultured cells are usually used for the production of biopharmaceuticals, and the amount of production per culture volume is limited.
- biopharmaceuticals are produced by connecting the cDNA of the target protein (antibody, bioactive peptide, etc.) downstream of a strong promoter represented by the CMV promoter, and expressing it in cultured mammalian cells such as HEK and CHO.
- a strong promoter represented by the CMV promoter represented by the CMV promoter
- biopharmaceuticals is determined by the protein-producing capacity of cells, the culture volume and the culture time, it is necessary to increase the protein-producing capacity of cells in order to increase production while suppressing production costs.
- the purpose of the present disclosure is to increase the yield of the target protein in protein production using cultured cells.
- the present inventors have found that activating PKC can enhance the transcriptional activity of promoters such as the CMV promoter, and have found a new method for activating PKC, thereby producing the desired protein in cultured cells. It was revealed that the amount could be increased.
- the present disclosure provides a method for producing a protein of interest operably linked to a promoter comprising a binding site for at least one transcription factor selected from SP1, CEBP, AP1, NF- ⁇ B and YY1.
- culturing a cell containing a nucleic acid encoding a linked protein of interest under conditions that activate PKC A method is provided wherein culturing under conditions that activate PKC is any of the following: (1) culturing said cells in the presence of a PKC activator and a calmodulin inhibitor; (2) expressing activated PKC in the cells and culturing in the presence of a calmodulin inhibitor; (3) expressing a nucleic acid encoding a peptide containing a Pro region or a RING region in said cells and culturing in the presence of a PKC activator; (4) The cells are cultured in the presence of a compound selected from or an ester, salt or solvate thereof.
- the present disclosure provides a method of enhancing transcription of a gene of interest operable on a promoter comprising a binding site for at least one transcription factor selected from SP1, CEBP, AP1, NF- ⁇ B and YY1.
- culturing under conditions that activate PKC a cell containing the nucleic acid of the gene linked to A method wherein culturing under conditions that activate PKC is any of the following: (1) culturing said cells in the presence of a PKC activator and a calmodulin inhibitor; (2) expressing activated PKC in the cells and culturing in the presence of a calmodulin inhibitor; (3) expressing a nucleic acid encoding a peptide containing a Pro region or a RING region in said cells and culturing in the presence of a PKC activator; (4) culturing the cells in the presence of compound X and a compound selected from compounds #1 to #7 or an ester, salt or solvate thereof;
- the present application provides a kit for producing a protein of interest, comprising: (1) PKC activators and calmodulin inhibitors, (2) a nucleic acid encoding activated PKC and a calmodulin inhibitor; (3) a nucleic acid encoding a peptide comprising a PKC activator and a Pro region or a RING region, or (4) a compound selected from compound X and compounds #1 to #7 or an ester, salt or solvate thereof; including Production of the protein of interest comprises a cell comprising a nucleic acid encoding the protein of interest operably linked to a promoter comprising a binding site for at least one transcription factor selected from SP1, CEBP, AP1, NF- ⁇ B and YY1.
- a kit is provided, comprising culturing.
- the present application provides a promoter comprising a binding site for at least one transcription factor selected from SP1, CEBP, AP1, NF- ⁇ B and YY1, operably linked, a nucleic acid encoding a protein, an internal ribosome Expression constructs are provided that include an entry site (IRES) or 2A self-cleaving peptide (2A peptide) sequence and a nucleic acid encoding activated PKC.
- a promoter comprising a binding site for at least one transcription factor selected from SP1, CEBP, AP1, NF- ⁇ B and YY1, operably linked, a nucleic acid encoding a protein, an internal ribosome Expression constructs are provided that include an entry site (IRES) or 2A self-cleaving peptide (2A peptide) sequence and a nucleic acid encoding activated PKC.
- IRS entry site
- 2A peptide 2A self-cleaving peptide
- the present application provides, operably linked, a promoter comprising a binding site for at least one transcription factor selected from SP1, CEBP, AP1, NF- ⁇ B and YY1, a nucleic acid encoding a protein, and
- An expression construct is provided that includes a nucleic acid encoding a peptide that includes a Pro region or a RING region.
- the present application provides a composition for activating PKC comprising Compound X and a compound selected from Compounds #1-#7 or an ester, salt or solvate thereof.
- the present disclosure can increase the production of the target protein.
- Luciferase activity in HEK293A cells constitutively expressing pRL-CMV in the presence or absence of PKC inhibitors in the presence of either compound X or compounds #1-#7.
- Figure 2 shows changes in luciferase activity in the presence or absence of Compound X in HEK293A cells constitutively expressing pRL-CMV (Renilla Luciferase).
- Luciferase activity in HEK293A cells constitutively expressing pRL-CMV (Renilla Luciferase) in the presence or absence of PKC inhibitors in the presence of either compound X or PKC activators #1-#4 indicates Luciferase activity in HEK293A cells constitutively expressing pRL-CMV (Renilla Luciferase) in the presence or absence of PKC inhibitors in the presence of either compound X or PKC activators #5-#6 indicates Luciferase activity in HEK293A cells transfected with pRL-CAG or pRL-EF1 in the presence of either compound X or compounds #1-#5 in the presence or absence of PKC inhibitors.
- Luciferase in pRL-CAG-transfected HEK293A cells in the presence or absence of PKC inhibitors in the presence of either compound X, compounds #6-#7 and PKC activators #1-#6 It shows activity. Luciferase in pRL-EF1 transfected HEK293A cells in the presence or absence of PKC inhibitors in the presence of either compound X, compounds #6-#7 and PKC activators #1-#6 It shows activity. PKC levels in HEK293A cells in the presence or absence of PKC inhibitors in the presence of any of Compound X, Compounds #1-#7, PKC activators #1-#6, Compound SC and Compound SB The phosphorylation state of substrate proteins is shown.
- Figure 2 shows luciferase activity in HEK293A cells constitutively expressing pRL-CMV (Renilla Luciferase) in the presence of compound X and/or compound SC and in the presence or absence of PKC inhibitors.
- Figure 2 shows luciferase activity in HEK293A cells constitutively expressing pRL-CMV (Renilla Luciferase) in the presence of compound X, compound SC and/or SB.
- FIG. 2 shows luciferase activity in HEK293A cells constitutively expressing pRL-CMV (Renilla Luciferase) in the presence of compound X or compound X+compound SC and in the presence of various histone deacetylase inhibitors.
- HEK293A cells were transfected with an expression construct in which a mouse IgG antibody heavy chain cDNA was linked under the control of a CMV promoter and an expression construct in which a mouse IgG antibody light chain cDNA was linked under the control of a CMV promoter, and compound X and compound # 1 to #5 and PKC activators #1 to #6, cultured in the presence or absence of compound SB+SC, and the concentration of mouse IgG antibody in the medium was measured. .
- HEK293A cells constitutively expressing human proinsulin under the control of the CMV promoter were incubated in medium supplemented with compound X, compounds #1-#5, PKC activators #1-#3, #5 or #6.
- HEK293A cells were transfected with an expression construct in which human leptin cDNA was linked under the control of the CMV promoter, and either compound X, compounds #1 to #5, or PKC activators #1 to #6 were added in medium.
- the results of culturing in the presence or absence of compound SB+SC and measuring the concentration of human leptin in the medium are shown.
- HEK293A cells which constitutively express mouse IgG antibody heavy and light chains, human proinsulin or human leptin under the control of the CMV promoter, were cultured in the presence or absence of compound X or compound X+SB, and each Results of measuring protein concentration over 3 or 4 days are shown.
- FIG. 4 shows the results of measuring luciferase activity after transfecting HEK293A cells with an expression construct in which a CMV promoter, Rluc cDNA, IRES and activated PKC cDNA are linked.
- the result of transfecting HEK293A cells with expression constructs in which the CMV promoter, Rluc cDNA, IRES or P2A, and activated PKC cDNA were linked, and measuring the luciferase activity is shown.
- An expression construct linking the CMV promoter, mouse IgG antibody heavy chain cDNA, IRES and activated PKC cDNA and an expression construct linking the CMV promoter, mouse IgG antibody light chain cDNA, IRES and activated PKC cDNA were prepared.
- HEK293A cells were transfected with an expression construct in which the CMV promoter, human leptin cDNA, IRES and activated PKC cDNA were linked, and the concentration of leptin in the medium was measured for 4 days.
- Figure 2 shows luciferase activity in the presence of naphthalenesulfonamide derivatives, in the presence or absence of compound X, in HEK293A cells transfected with pRL-CMV. Schematic representation of expression constructs linking the CMV promoter, Rluc cDNA, IRES and PML (wild-type or deleted) cDNA.
- Figure 2 shows luciferase activity in the presence or absence of compound X in HEK293A cells transfected with expression constructs containing CMV promoter, Rluc cDNA, IRES and PML (wild-type or deleted) cDNA.
- Luciferase activity in the presence or absence of Compound X, SB, Compound SC or combinations thereof in HEK293A cells transfected with expression constructs containing CMV promoter, Rluc cDNA, IRES and PML ⁇ 9 or PML-Ring cDNA indicates BLAST analysis of the amino acid sequence of the RING region of PML is performed, and a phylogenetic tree among genes having homologous amino acid sequences is shown.
- FIG. 3 shows luciferase activity in the presence or absence of compound X in HEK293A cells transfected with an expression construct linking the CMV promoter, Rluc cDNA, IRES and RING region cDNAs.
- FIG. 3 shows luciferase activity in the presence or absence of compound X in HEK293A cells transfected with an expression construct linking the CMV promoter, Rluc cDNA, IRES and RING region cDNAs.
- Schematic diagram of an expression construct in which the CMV promoter, Rluc cDNA, IRES and PML (stop codon introduction) cDNAs are linked and an expression construct in which the CMV promoter, Rluc cDNA and PML (wild-type or deleted) cDNA are linked.
- the method for producing the protein of the present disclosure utilizes genetic engineering techniques in which a nucleic acid encoding a target protein is introduced into cultured cells, and the protein is expressed and recovered.
- genetic engineering techniques are well known in the art and can be carried out according to methods described in literature (for example, Molecular Cloning, T. Maniatis et al., CSH Laboratory (1983), DNA Cloning, D.M. Glover, IRL PRESS (1985)).
- the promoter may contain a binding site for at least one transcription factor selected from SP1, CEBP, AP1, NF- ⁇ B and YY1.
- the promoter may also contain binding sites for other transcription factors, such as CREB.
- the promoter comprises binding sites for SP1, CEBP, AP1, NF- ⁇ B and YY1.
- the promoter comprises binding sites for SP1, CEBP, AP1, NF- ⁇ B, YY1 and CREB.
- promoters that may be used include the CMV promoter, the CAG promoter and the EF1 promoter, especially the CMV promoter.
- operably linked means that the regulatory sequence elements, such as the promoter, IRES and 2A peptide sequences, and the nucleic acid encoding the protein are linked in a manner that allows expression of the protein. meaning that the 3' end of each DNA and the 5' end of its downstream DNA may be directly linked, and any DNA sequence may exist between them.
- the target protein is not limited as long as it does not have unacceptable toxicity to the cultured cells used.
- the protein of interest can be the active ingredient of a biopharmaceutical such as immunoglobulin, leptin, insulin or fragments thereof.
- a nucleic acid encoding a protein of interest can be produced based on the amino acid sequence information of the protein and the sequence information of the nucleic acid encoding it, for example, by conventional DNA synthesis or amplification by RT-PCR.
- An expression construct can be created by incorporating a promoter and a nucleic acid encoding the target protein into an expression vector.
- the expression vector used here can be appropriately selected according to the host to be used, the purpose, etc. Examples thereof include plasmids, phage vectors, virus vectors, and the like. Examples include plasmid vectors such as pKCR, pCDM8, pGL2, pcDNA3.1, pRc/RSV and pRc/CMV, and viral vectors such as retroviral vectors, adenoviral vectors and adeno-associated viral vectors.
- the vector may optionally have elements such as a selection marker gene and a terminator.
- Host cells are typically animal cells such as HEK293A cells, HEK293T cells, CHO cells, COS cells, Vero cells, HeLa cells, L929 cells, BALB/c3T3 cells, C127 cells and the like.
- a normal introduction method suitable for the host cell may be used. Specific examples include calcium phosphate method, DEAE-dextran method, electroporation method, lipofection method and the like.
- the transformed cells thus obtained may transiently express the protein of interest, or a cell line stably expressing the protein of interest may be established.
- the target protein By culturing transformed cells under conditions that activate PKC, the target protein can be produced efficiently.
- Culture conditions such as medium, culture time, and culture temperature suitable for culturing each cell are well known to those skilled in the art, and may be appropriately selected. For example, under conditions that activate PKC, 1 hour or more, 2 hours or more, 4 hours or more, 6 hours or more, 8 hours or more, 12 hours or more, 18 hours or more, 24 hours or more, 48 hours or more, and 60
- the protein of interest is recovered within hours, within 48 hours, within 36 hours, within 30 hours or within 24 hours after culturing the cells.
- the resulting protein can be further isolated and purified by common biochemical purification means. Examples of purification means include salting out, ion exchange chromatography, adsorption chromatography, affinity chromatography, gel filtration chromatography and the like.
- the conditions for activating PKC are (1) culturing the cells in the presence of a PKC activator and a calmodulin inhibitor.
- PKC is a type of protein kinase that phosphorylates the hydroxyl groups of serine and threonine residues of substrate proteins, and more than 10 isoenzymes are known. Isozymes are classified into conventional ( ⁇ , ⁇ I, ⁇ II, ⁇ ), novel ( ⁇ , ⁇ , ⁇ , ⁇ ) and atypical ( ⁇ , M ⁇ , ⁇ / ⁇ ) types depending on their structures, activation mechanisms, and physiological activities. It is classified into three subfamilies. In the present disclosure, PKC is preferably a conventional PKC isozyme or a new PKC isozyme, particularly preferably PKC ⁇ or PKC ⁇ .
- PKC activator means a substance that enhances the kinase activity of PKC. Two or more PKC activators may be used in combination.
- the PKC activator is of formula (I): ⁇ In the formula, R 1 is H, halogen, —OH, C 1-6 alkyl, C 2-6 alkenyl, C 2-6 alkynyl, C 1-6 alkoxy, C 6-14 aryl or —OC(O)R 3 , wherein C 1-6 alkyl, C 2-6 alkenyl, C 2-6 alkynyl, C 1-6 alkoxy or aryl are optionally substituted by 1 to 3 halogens, the same or different, R 2 is C 6-12 alkyl, C 6-12 alkenyl, C 6-12 alkynyl or C 6-12 alkoxy, where C 6-12 alkyl, C 6-12 alkenyl, C 6-12 alkynyl or C 6-12 alkoxy is optionally substituted by 1 to 3 halogens, which may be the same or different; R 3 is C 1-6 alkyl, C 2-6 alkenyl, C 2-6 alkynyl, amino or C
- halogen means an atom selected from fluorine, chlorine, bromine and iodine.
- alkyl denotes a saturated, straight or branched chain hydrocarbon group.
- alkenyl means a straight or branched chain hydrocarbon containing one or more double bonds.
- alkynyl means a straight or branched chain hydrocarbon containing one or more triple bonds.
- alkoxy means -O-alkyl, where alkyl is defined in this disclosure.
- aryl means a monovalent aromatic carbocyclic group of 6 to 14 carbon atoms having a single ring (eg phenyl) or multiple condensed rings (eg naphthyl or anthryl). do.
- Aryl typically includes phenyl and naphthyl.
- amino means the group -NH2 .
- ester means an ester that can be hydrolyzed in vivo or in vitro, including those that are readily degraded to release the parent compound or a salt thereof.
- Suitable ester groups are, for example, those derived from aliphatic carboxylic acids, especially alkanoic acids, alkenoic acids, cycloalkanoic acids and alkanedioic acids, wherein each alkyl or alkenyl group has, for example, up to 6 carbon atoms. have).
- specific esters include formates, acetates, propionates, butyrates, acrylates and ethylsuccinates.
- a “salt” may be a salt of a compound with an inorganic or organic acid.
- Preferred salts are those with inorganic acids such as hydrochloric, hydrobromic, phosphoric or sulfuric acids, or with organic carboxylic or sulfonic acids such as acetic acid, trifluoroacetic acid, propionic acid, maleic acid, fumaric acid, It is a salt with malic acid, citric acid, tartaric acid, lactic acid, benzoic acid or methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, toluenesulfonic acid, naphthalenesulfonic acid or naphthalenedisulfonic acid.
- Salts can also be salts with customary bases, such as alkali metal salts (for example sodium or potassium salts), alkaline earth metal salts (for example calcium or magnesium salts), or ammonia or organic amines (for example diethylamine, triethylamine, ammonium derived from ethyldiisopropylamine, procaine, dibenzylamine, N-methylmorpholine, dihydroabiethylamine, methylpiperidine, L-arginine, creatine, choline, L-lysine, ethylenediamine, benzathine, ethanolamine, meglumine or tromethamine) It may be a salt, especially the sodium salt.
- alkali metal salts for example sodium or potassium salts
- alkaline earth metal salts for example calcium or magnesium salts
- ammonia or organic amines for example diethylamine, triethylamine, ammonium derived from ethyldiisopropyl
- solvate means a compound that forms a complex through coordination with solvent molecules in a solid or liquid state.
- Preferred solvates are hydrates.
- R 1 is H or —OC(O)R 3 ;
- R 2 is C 6-12 alkyl or C 6-12 alkenyl,
- R 3 is C 1-6 alkyl or C 6-14 aryl.
- R 1 is H or —OC(O)R 3 ;
- R 2 is nonyl or 1,3-nonadienyl, R3 is methyl or phenyl.
- the compound of formula (I) is compound X, compound #1 or compound #2, described below.
- R 4 is H or —OC(O)R 6 ;
- R 5 is C 1-6 alkyl or C 2-6 alkenyl;
- R 6 is C 1-18 alkyl.
- R 4 is H or —OC(O)R 6 ;
- R5 is methyl, propyl, sec-butyl or butenyl;
- R6 is propyl, nonyl or tridecyl.
- the compound of formula (II) is Compound #3, Compound #4, Compound #5, TPA, phorbol 12,13-dibutyrate or prostratin, particularly Compound #3, Compound #4 or Compound # 5.
- R 7 is H or —C(O)R 9 ;
- R 8 is H or —C(O)R 9 ;
- R 9 is C 1-18 alkyl or C 2-18 alkenyl.
- R 7 is H or —C(O)R 9 ;
- R 8 is H or —C(O)R 9 ;
- R9 is pentadecyl or butenyl.
- the compound of formula (III) is compound #6, compound #7 or ingenol 3-angelate, particularly compound #6 or compound #7, described below.
- Compounds of formulas (I)-(III) or their esters, salts or solvates are, for example, 0.1-10 ⁇ g/ml, 0.1-1 ⁇ g/ml, 0.1-1000 ng/ml, 2-500 ng /ml or 5-200 ng/ml to the culture medium.
- the PKC activator is a compound of formula (I) or an ester, salt or solvate thereof. These can be added to the culture medium in concentrations of, for example, 0.1-10 ⁇ g/ml, 0.1-1 ⁇ g/ml, 0.1-1000 ng/ml, 20-500 ng/ml or 50-200 ng/ml.
- the PKC activator is a compound selected from Compound X and Compounds #1-#7 below or an ester, salt or solvate thereof.
- the PKC activator is Compound X or an ester, salt or solvate thereof.
- PKC activator A known PKC activator may be used.
- PKC activators include 12-O-tetradecanoylphorbol 13-acetate (TPA, also called phorbol 12-myristate 13-acetate (PMA)), prostratin, bryostatin 1, bryostatin 2, FR236924 , ( ⁇ )-indolactam V, PEP005, phorbol 12,13-dibutyrate, 1-oleoyl-2-acetyl-sn-glycerol, 1-O-hexadecyl-2-O-arachidonyl-sn-glycerol, 1,2 -dioctanoyl-sn-glycerol, PIP2, resiniferatoxin, phorbol 12,13-dihexanoate, mezerein, ingenol 3-angelate, RHC-80267, DCP-LA, lipoxin A4, (2S,5S)-(E ,E)-8-(5-(4
- the PKC activator is TPA, prostratin, ( ⁇ )-indolactam V, phorbol 12,13-dibutyrate, ingenol 3-angelate, or (2S,5S)-(E,E)- 8-(5-(4-(trifluoromethyl)phenyl)-2,4-pentadienoylamino)benzolactam.
- Known PKC activators can be used as appropriate by methods known in the art, such as methods recommended by manufacturers.
- the PKC activator is Compound X, Compounds #1-#7, TPA, prostratin, ( ⁇ )-indolactam V, phorbol 12,13-dibutyrate, ingenol 3-angelate and (2S,5S )-(E,E)-8-(5-(4-(trifluoromethyl)phenyl)-2,4-pentadienoylamino)benzolactam or an ester, salt or solvate thereof; be.
- Calmodulin is an acidic protein that functions as a calcium sensor, regulating intracellular calcium levels.
- formula (IV) ⁇ In the formula, n is an integer from 1 to 8, R is C 1-6 alkyl, C 2-6 alkenyl, C 2-6 alkynyl, C 1-6 alkoxy, C 6-14 aryl, amino, hydroxy, COOH or COOR', where R' is C 1-6 alkyl ⁇ or an ester, salt or solvate thereof may be used.
- n is an integer from 4 to 6
- R is C 1-6 alkyl, C 6-14 aryl or amino.
- n is an integer of 4 or 6; R is methyl, phenyl or amino.
- the compounds of formula (IV) include, for example, SC-9, SC-10 and W-7.
- Compounds of formula (IV) may be used at concentrations of, for example, 0.5-100 ⁇ g/ml, 1-50 ⁇ g/ml or 2-10 ⁇ g/ml.
- calmodulin inhibitors such as W-7, calmidazolium, bisindolylmaleimide I, trifluoperazine, ruthenium red, ophiovorin A, CaM kinase II (290-309), E6 berbamine, mastoparan, compound 48/80, phenoxy Benzamine, W-7 isomer, Polystesmastoparan, A-7, Fluphenazine-N-2-chloroethane, W-13, W-13 isomer, CGS 9343B, W-5 isomer, W-12, N-(5 -aminopentyl)-5-chloro-2-naphthalenesulfonamide, W-5, may be used as appropriate by methods known in the art, such as manufacturer's recommendations. Two or more calmodulin inhibitors may be used in combination.
- the condition for activating PKC is (2) allowing the cell to express activated PKC and culturing in the presence of a calmodulin inhibitor.
- Activated PKC means a PKC mutant that constitutively exhibits kinase activity.
- Activated PKC can be PKC lacking the N-terminal regulatory region (Molecular and Cellular Biology 19(2):1313-24, 1999).
- Activated PKC may further have mutations that enhance kinase activity (PNAS 115(24):E5497-E5505. 2018).
- Examples of activated PKC include PKC ⁇ -CA (SEQ ID NO: 1) and PKC ⁇ CA-M489V (SEQ ID NO: 2).
- Kinase activity of a PKC mutant can be measured by various methods known in the art. For example, a method of overexpressing a PKC mutant in cultured cells and detecting the phosphorylation level of the substrate by Western blotting using a phosphorylated substrate-specific antibody (for example, THE JOURNAL OF BIOLOGICAL CHEMISTRY, Vol. 279, No. 27, pp. 27986-27993, 2004), ELISA (for example, Cell Death and Differentiation (2015) 22, 2078-2086), and in vitro incorporation of phosphate groups into substrates using 32 P-gamma-ATP. Evaluation methods (eg, THE JOURNAL OF BIOLOGICAL CHEMISTRY, Vol. 262, No. 20, pp. 9569-9573, 1987) and the like can be used. Various kits that measure kinase activity may be used, such as the PKC kinase activity kit (Enzo Life Science, #ADI-EKS-420A).
- cells expressing a PKC mutant are cultured in the presence and absence of a PKC inhibitor, and the amount of phosphorylated protein is determined by an immunological method using an antibody that specifically recognizes the phosphorylated protein.
- a PKC mutant can be determined to be an activated form of PKC if it is measured and the amount of phosphorylated protein is reduced by a PKC inhibitor.
- immunological techniques include flow cytometry analysis, radioisotope immunoassay (RIA), enzyme immunoassay (ELISA), western blotting, and immunohistochemical staining.
- expression comprising, operably linked, a promoter, a nucleic acid encoding a protein of interest, an internal ribosome entry site (IRES) or 2A self-cleaving peptide (2A peptide) sequence, and a nucleic acid encoding an activated PKC.
- Constructs can be used to express proteins of interest and activated PKC in cells.
- a promoter, a nucleic acid encoding a protein of interest, an IRES or 2A peptide sequence, and a nucleic acid encoding activated PKC are operably linked in this order.
- IRES is an RNA region that can recruit eukaryotic ribosomes to mRNA, allowing cap-independent initiation of translation as part of the process of protein synthesis.
- Many IRESs have been identified in viral and eukaryotic genomes, and synthetic IRESs have also been developed.
- IRES can be identified by enteroviruses (e.g. human papillomavirus 1, human coxsackievirus B); rhinoviruses (e.g. human rhinovirus); hepatoviruses (hepatitis A virus); cardioviruses (encephalomyocarditis virus ECMV and Theiler's encephalomyelitis virus) ); aphthoviruses (foot-and-mouth disease virus, equine rhinitis A virus, equine rhinitis B virus); pestiviruses (e.g. bovine viral diarrhea virus and swine fever virus; hepaciviruses (e.g.
- enteroviruses e.g. human papillomavirus 1, human coxsackievirus B
- rhinoviruses e.g. human rhinovirus
- hepatoviruses hepatoviruses (hepatitis A virus)
- cardioviruses encephalomyocardit
- the IRES is a virus of the retroviridae family, such as members of the lentivirus family (e.g., simian immunodeficiency virus and human immunodeficiency virus 1); BLV-HTLV retroviruses (e.g., human T-lymphotropic virus type 1); ); and mammalian C-type retrovirus families (e.g., Moloney murine leukemia virus, Friend murine leukemia virus, Harvey murine sarcoma virus, avian reticuloendotheliosis virus, murine leukemia virus (envRNA), Rous sarcoma virus).
- retroviridae family such as members of the lentivirus family (e.g., simian immunodeficiency virus and human immunodeficiency virus 1); BLV-HTLV retroviruses (e.g., human T-lymphotropic virus type 1); ); and mammalian C-type retrovirus families (e.g., Moloney
- IRESs derived from eukaryotic mRNAs include, for example, the IRESs of BiP, Drosophila Antennapedia (exons d and e), c-myc, and the X-linked inhibitor of apoptosis (XIAP) gene.
- IRESs Various synthetic IRESs have also been developed, for example De Gregorio et al. (1999) EMBO J. 75:4865-74; Owens et al. (2001) PNAS 4:1471-6; and Venkatesan et al. (2001) Molecular and Cellular Biology 21:2826-37.
- IRESs known in the art see, for example, rangueil.inserm.fr/IRESdatabase.
- an IRES from encephalomyocarditis virus ECMV is used.
- the 2A peptide sequence induces ribosome skipping during protein translation.
- a 2A peptide sequence is present in the amino acid sequence of a protein, the protein is translated as two polypeptides truncated at the C-terminus of the 2A peptide sequence.
- 2A peptides include, for example, peptides described in Kim, J. H., et al., PLoS One.
- P2A peptide SEQ ID NO: 3: (GSG )ATNFSLLKQAGDVEENPGP
- T2A peptide SEQ ID NO: 4
- E2A peptide SEQ ID NO: 5: (GSG)QCTNYALLKLAGDVESNPGP
- F2A peptide SEQ ID NO: 6: (GSG)VKQTLNFDLLKLAGDVESNPGP
- the N-terminal GSG may or may not be present in each sequence).
- calmodulin inhibitors can be used in the same manner as in (1) above.
- the condition for activating PKC is (3) allowing the cell to express a nucleic acid encoding a peptide containing a Pro region or a RING region and culturing in the presence of a PKC activator.
- the target protein can be efficiently produced by using an expression construct containing a nucleic acid encoding the target protein and a nucleic acid encoding a peptide containing the Pro region or the RING region.
- an expression construct comprising, operably linked, a promoter, nucleic acid encoding a protein, and nucleic acid encoding a peptide comprising a Pro region or a RING region may be used.
- a promoter, a protein-encoding nucleic acid, and a peptide-encoding nucleic acid comprising a Pro region or a RING region are operably linked in this order.
- a nucleic acid encoding a peptide containing a Pro region or a RING region may or may not be translated into a peptide.
- an IRES or 2A peptide sequence may be inserted between the nucleic acid encoding the protein of interest and the nucleic acid encoding the peptide containing the Pro region or RING region.
- IRES and 2A peptide sequences can be used as in (2) above.
- an expression construct comprising, operably linked, a promoter, a nucleic acid encoding a protein, an IRES or 2A peptide sequence, and a nucleic acid encoding a peptide comprising a Pro region or a RING region may be used.
- a promoter, a nucleic acid encoding a protein, an IRES or 2A peptide sequence, and a nucleic acid encoding a peptide comprising a Pro or RING region are operably linked in that order.
- the Pro region or RING region may be of any species, e.g., mouse, rat, hamster, rabbit, cat, dog, bovine, porcine, ovine, monkey, human, etc., particularly human. It is. Preferably, the Pro region or RING region is from the same species as the cells used.
- a Pro region refers to a region having an amino acid sequence rich in proline residues and mediates specific interactions with functional domains such as WW domains and SH3 domains.
- Pro regions include repetitive short proline residue-rich amino acid sequences, tandemly repeated proline residue-rich amino acid sequences, non-repetitive proline residue-rich amino acid sequences, and hydroxyproline residues.
- regions with amino acid sequences rich in Pro regions include, but are not limited to, integral membrane proteins such as nuclear proteins, transcription factors, transporters, channels and receptors, globular proteins, hormones, neuropeptides, mucins, immunoglobulins, and extracellular matrix proteins. can be found in a variety of proteins, including
- the Pro region may be derived from any protein. Proteins containing the Pro region include, for example, PML, ARHGEF1, aggrecan-1, RALGDS, DGKK, SPATA21, Rabufilin-3A, TEAD3, SPPL2B and FLJ43093.
- the RING region also called the RING finger region, binds to a pair of zinc atoms and mediates protein-protein interactions.
- RING regions generally have the following consensus sequences.
- C is a cysteine residue
- H is a histidine residue
- X is any amino acid residue.
- the RING region may be derived from any protein.
- proteins containing a RING region include TRIM13, LONRF3, TRIM47, RNF135, TRIM10, TRIM72, TRIM60, TRIM39, TRIM4, TRIM43B, TRIM43, TRIM25, TRIM26, TRIM31, HTLF, BRCA1, TRIM50, TRIM21, SSA1, TRIM5d, TRIM22 , KIAA0182, TRIM65, RAG1, BFAR, Pex10, RNF8, RING2, COPI, TRIM2, TRIM3, SH3RF2, PML and TRIM56.
- the RING region is the RING region of PML, TRIM3, TRIM56, COPI, Pex10, BRCA1 or HTLF.
- the Pro region or RING region is derived from promyelocytic leukemia protein (PML).
- PML is required for the assembly of intranuclear structures called PML bodies.
- PML bodies have diverse functions and have been suggested to be involved in a wide range of intracellular processes.
- a plurality of isoforms are known for human PML, and the N-terminal side has the same amino acid sequence in all isoforms.
- the amino acid and nucleotide sequences of human wild-type (WT) PML isoform 5 (Gene ID: 5371, NCBI Reference Sequence: NP_150247.2) are shown in SEQ ID NOs: 7 and 8.
- the amino acid sequence consists of 560 amino acids and has each region shown in FIG.
- positions 1 to 45 (SEQ ID NO: 9) are the Pro region
- positions 46 to 105 (SEQ ID NO: 10) are the RING region.
- Positions 1 to 135 (SEQ ID NO: 11) of the nucleotide sequence of SEQ ID NO: 8 encode the Pro region
- positions 136 to 315 (SEQ ID NO: 12) encode the RING region.
- the Pro region corresponds to the region of positions 1 to 45 of SEQ ID NO: 7. It can be the region of the protein that corresponds.
- the Pro region comprises or consists of an amino acid sequence having at least 90% or more identity to the amino acid sequence of SEQ ID NO:9.
- the Pro region comprises the amino acid sequence of SEQ ID NO:9.
- the Pro region consists of the amino acid sequence of SEQ ID NO:9.
- the Pro region is encoded by a nucleotide sequence having at least 90% or more identity to the nucleotide sequence of SEQ ID NO:11 or comprising it. In some embodiments, the Pro region is encoded by a nucleotide sequence comprising the nucleotide sequence of SEQ ID NO:11. In one embodiment, the Pro region is encoded by the nucleotide sequence of SEQ ID NO:11.
- the RING region can be a region of a protein that corresponds to the region from position 46 to position 105 of SEQ ID NO: 7 when the amino acid sequence of a protein and the amino acid sequence of SEQ ID NO: 7 are optimally aligned. .
- the RING region comprises or consists of an amino acid sequence having at least 90% or more identity to the amino acid sequence of SEQ ID NO:10.
- the RING region comprises the amino acid sequence of SEQ ID NO:10.
- the RING region consists of the amino acid sequence of SEQ ID NO:10.
- the RING region is encoded by a nucleotide sequence having at least 90% or more identity to the nucleotide sequence of SEQ ID NO:12, or by a nucleotide sequence comprising the same. In some embodiments, the RING region is encoded by a nucleotide sequence comprising the nucleotide sequence of SEQ ID NO:12. In one embodiment, the RING region is encoded by the nucleotide sequence of SEQ ID NO:12.
- nucleotide or amino acid sequence identity refers to the degree of sequence similarity between nucleic acids or proteins, and is optimal (maximum nucleotide or amino acid identity) over the region of the sequences to be compared. ) is determined by comparing the two sequences aligned to . A numerical value (%) of sequence identity is determined by determining the number of identical nucleotides or amino acids present in both sequences to determine the number of matching sites, and then dividing this number of matching sites by nucleotides or amino acids within the sequence region being compared. It is calculated by dividing by the total number and multiplying the resulting number by 100.
- Algorithms for obtaining optimal alignment and sequence identity include various algorithms commonly available to those of skill in the art (eg, BLAST algorithms, FASTA algorithms, etc.). Sequence identity can be determined, for example, using sequence analysis software such as BLAST, FASTA.
- the PKC activator can be used in the same manner as in (1) above.
- the condition for activating PKC is (4) culturing the cells in the presence of compound X and a compound selected from compounds #1 to #7 or esters, salts or solvates thereof. be. These compounds are included in the PKC activators described in (1) above and can be used as described above.
- Compound X and compounds #1 to #7 may be obtained by chemical synthesis or may be extracted from plants containing them.
- compound X is from Lowdaphne Stringbush
- compounds #1 and #2 are from Lilac Daphne
- compounds #3-#5 are from Croton
- compounds #6-#7 are Caper Euphorbia).
- Plant products containing these compounds, such as plant extracts or extracts, may also be used.
- a composition for activating PKC comprising a compound selected from Compound X and Compounds #1 to #7 or esters, salts or solvates thereof.
- the composition may contain, for example, suitable carriers, excipients, additives, etc., and may contain other active ingredients.
- a histone deacetylase inhibitor may be added to the medium when PKC is activated under the conditions (1) to (4).
- Histone deacetylase is an enzyme that deacetylates histones, which are major constituents in chromatin structure, and plays an important role in the regulation of gene transcription.
- histone deacetylase inhibitors such as trichostatin A, M344, butyrate, phenylbutyrate, apicidin, valproic acid, BML-210, depudecin, romidepsin (FK-228), HC toxin, oxamflatin, scriptaid, Splitomycin, suberoyl bis-hydroxamic acid, vorinostat, dacinostat (LAQ-824), panobinostat (LBH-589), belinostat (PXD-101), phenyl acetate, IF2357, FK-228, entinostat (MS-275), mosetinostat (MGCD0103) or tacedinaline (CI994), preferably sodium butyrate, valproic acid, trichostatin A, vorinostat, apicidin, entinostat or tacedinaline, particularly preferably sodium butyrate, by methods known in the art, e.g. can be used as appropriate according to the method
- a calmodulin inhibitor may be added to the medium when PKC is activated under conditions (3) or (4).
- a calmodulin inhibitor can be used in the same manner as in (1) above.
- the disclosure also provides a method of enhancing transcription of a gene of interest, wherein the promoter is operably linked to a promoter comprising binding sites for at least one transcription factor selected from SP1, CEBP, AP1, NF- ⁇ B and YY1.
- a method comprising culturing a cell containing the nucleic acid of the gene of interest under conditions that activate PKC. Cultivation under conditions that activate PKC is any of (1) to (4) described with respect to the protein production method.
- kits that can be used in the methods of the present disclosure.
- Each component contained in the kit may be dissolved in water or a suitable buffer, or lyophilized and contained in a suitable container, either separately or, if possible, in admixture.
- Suitable containers include bottles, vials, test tubes, tubes, plates and the like.
- the container may be made from a variety of materials such as glass, plastic, metal, and the like.
- the present inventors have found that the transcriptional activity of the CMV promoter can be enhanced by expressing a nucleic acid encoding a peptide containing a Pro region or RING region. Accordingly, in one aspect, the present application provides a method for producing a protein of interest, wherein the binding site for at least one transcription factor selected from SP1, CEBP, AP1, NF- ⁇ B and YY1 is operably linked , a nucleic acid encoding a protein of interest, and a nucleic acid encoding a peptide comprising a Pro region or a RING region. This method can be performed according to (3) above, but without using a PKC activator.
- a method for producing a protein of interest which encodes the protein of interest operably linked to a promoter containing a binding site for at least one transcription factor selected from SP1, CEBP, AP1, NF- ⁇ B and YY1 culturing a cell containing a nucleic acid that activates PKC
- a method, wherein culturing under conditions that activate PKC is any of the following: (1) culturing said cells in the presence of a PKC activator and a calmodulin inhibitor; (2) expressing activated PKC in the cells and culturing in the presence of a calmodulin inhibitor; (3) expressing a nucleic acid encoding a peptide containing a Pro region or a RING region in said cells and culturing in the presence of a PKC activator; (4) culturing the cells in the presence of compound X and a compound selected from compounds #1 to #7 or an ester, salt or solvate thereof; [2] The
- a method for enhancing transcription of a gene of interest wherein the gene is operably linked to a promoter comprising a binding site for at least one transcription factor selected from SP1, CEBP, AP1, NF- ⁇ B and YY1 culturing a cell containing the nucleic acid of A method, wherein culturing under conditions that activate PKC is any of the following: (1) culturing said cells in the presence of a PKC activator and a calmodulin inhibitor; (2) expressing activated PKC in the cells and culturing in the presence of a calmodulin inhibitor; (3) expressing a nucleic acid encoding a peptide containing a Pro region or a RING region in said cells and culturing in the presence of a PKC activator; (4) culturing the cells in the presence of compound X and a compound selected from compounds #1 to #7 or an ester, salt or solvate thereof;
- any one of items 1 to 7, wherein the culturing under conditions that activate PKC is (1) culturing the cells in the presence of a PKC activator and a calmodulin inhibitor. described method.
- the culture under conditions that activate PKC is (2) allowing the cells to express activated PKC and culturing in the presence of a calmodulin inhibitor. Any method described.
- the cell comprises an expression construct comprising, operably linked, a promoter, a nucleic acid encoding a protein of interest, an internal ribosome entry site or 2A peptide sequence, and a nucleic acid encoding an activated PKC. , paragraph 9.
- Culturing under conditions that activate PKC is (3) allowing cells to express a nucleic acid encoding a peptide containing a Pro region or a RING region, and culturing in the presence of a PKC activator. 8. The method according to any one of items 1 to 7. [14] The method according to item 13, wherein a nucleic acid encoding a peptide containing a RING region is expressed in a cell.
- the RING area is CX 2 -CX 9-39 -CX 1-3 -HX 2-3 -CX 2 -CX 4-48- CX 2 -C ⁇ Wherein, C is a cysteine residue, H is a histidine residue, and X is any amino acid residue. ⁇ 15.
- the method of paragraph 14, comprising the amino acid sequence of [16]
- the RING region is TRIM13, LONRF3, TRIM47, RNF135, TRIM10, TRIM72, TRIM60, TRIM39, TRIM4, TRIM43B, TRIM43, TRIM25, TRIM26, TRIM31, HTLF, BRCA1, TRIM50, TRIM21, SSA1, TRIM5d, TRIM22, KIAA0182 , TRIM65, RAG1, BFAR, Pex10, RNF8, RING2, COPI, TRIM2, TRIM3, SH3RF2, PML or TRIM56.
- nucleic acid encoding the peptide containing the Pro region consists of a nucleotide sequence having at least 90% identity with the nucleotide sequence of SEQ ID NO: 11. .
- nucleic acid encoding a peptide containing a Pro region and a RING region is expressed in a cell.
- the cell comprises an expression construct comprising, operably linked, a promoter, a nucleic acid encoding a protein of interest, and a nucleic acid encoding a peptide comprising a Pro region or a RING region.
- a promoter, a nucleic acid encoding a protein of interest, and a nucleic acid encoding a peptide containing a Pro region or a RING region are operably linked in this order.
- the expression construct further comprises a nucleic acid encoding an internal ribosome entry site or 2A peptide sequence.
- a promoter, a nucleic acid encoding a protein of interest, an internal ribosome entry site or 2A peptide sequence, and a nucleic acid encoding a peptide containing a Pro region or RING region are operably linked in this order. 29. The method according to any one of paragraphs 27-29. [31] The method according to any one of items 13 to 30, wherein the cell is cultured in the presence of a calmodulin inhibitor.
- the PKC activator is compound X, compounds #1-#7, TPA, prostratin, (-)-indolactam V, phorbol 12,13-dibutyrate, ingenol 3-angelate and (2S,5S)- (E,E)-8-(5-(4-(trifluoromethyl)phenyl)-2,4-pentadienoylamino)benzolactam or an ester, salt or solvate thereof; 32.
- culturing under conditions that activate PKC comprises (4) culturing said cells in the presence of a compound selected from compound X and compounds #1 to #7 or an ester, salt or solvate thereof 8.
- a compound selected from compound X and compounds #1 to #7 or an ester, salt or solvate thereof 8.
- the method of paragraph 45 or 46, wherein the cell is cultured in the presence of a calmodulin inhibitor.
- the calmodulin inhibitor is a compound of formula (IV) or an ester, salt or solvate thereof.
- a kit for producing a protein of interest comprising: (1) PKC activators and calmodulin inhibitors, (2) a nucleic acid encoding activated PKC and a calmodulin inhibitor; (3) a nucleic acid encoding a peptide comprising a PKC activator and a Pro region or a RING region, or (4) a compound selected from compound X and compounds #1 to #7 or an ester, salt or solvate thereof; including Production of the protein of interest comprises a cell comprising a nucleic acid encoding the protein of interest operably linked to a promoter comprising a binding site for at least one transcription factor selected from SP1, CEBP, AP1, NF- ⁇ B and YY1.
- a kit comprising culturing.
- a promoter comprising a binding site for at least one transcription factor selected from SP1, CEBP, AP1, NF- ⁇ B and YY1, operably linked, a nucleic acid encoding a protein, an internal ribosome entry site or 2A
- An expression construct comprising a peptide sequence and a nucleic acid encoding activated PKC.
- Paragraph 55 wherein a promoter, a nucleic acid encoding a protein of interest, an internal ribosome entry site or 2A peptide sequence, and a nucleic acid encoding activated PKC are operably linked in this order. expression construct.
- a promoter comprising a binding site for at least one transcription factor selected from SP1, CEBP, AP1, NF- ⁇ B and YY1, operably linked, a nucleic acid encoding a protein, and a Pro region or RING
- An expression construct comprising a nucleic acid encoding a peptide containing region.
- paragraph 57 wherein a promoter, a protein-encoding nucleic acid, and a peptide-encoding nucleic acid comprising a Pro region or a RING region are operably linked in this order.
- the expression construct of paragraphs 57 or 58 comprising a nucleic acid encoding an internal ribosome entry site or 2A peptide sequence.
- a promoter, a nucleic acid encoding a protein of interest, an internal ribosome entry site or 2A peptide sequence, and a nucleic acid encoding a peptide containing a Pro region or RING region are operably linked in this order, 59.
- a composition for activating PKC comprising compound X and a compound selected from compounds #1 to #7 or an ester, salt or solvate thereof.
- pRL-CMV Promega, #E2261
- pCAGGS vector As the CAG promoter
- pEFBOS vector As the EF1 promoter
- a construct was prepared in which the CMV region of pRL-CMV was replaced.
- Mouse IgG antibody (heavy chain, light chain), human proinsulin and human leptin expression constructs were prepared by inserting these cDNAs downstream of the CMV promoter of a pcDNA vector.
- HEK293A cells human embryonic kidney cells
- DMEM medium containing 10% FCS and 1% penicillin/streptomycin under conditions of 37°C, 5% CO 2 and 90% humidity.
- a HEK293A cell line that constitutively expresses mouse IgG antibody, human proinsulin, and leptin was transfected with constructs that inserted these cDNAs downstream of the CMV promoter, followed by long-term culture to form single clones. . Thereafter, the expression of the target protein was confirmed from each clone using the ELISA method, and cell lines were established. These cell lines were also cultured under similar conditions.
- Transfected HEK293A cells were seeded in a 24-well plate at 1.0 ⁇ 10 5 cells/well and cultured for 24 hours. Unless otherwise specified, 50 ng of expression construct, 1 ⁇ l-Plus Reagent, and 1 ⁇ l-Lipofectamine LTX per well were prepared in 50 ⁇ l of Opti-MEM, added to each well of a 24-well plate in which cells were cultured, and incubated at 37°C for 24 hours. Transfection was performed by culturing.
- the medium was removed from the wells, medium supplemented with each concentration of compound was added, and cultured for an additional 24 hours. Thereafter, the medium was removed, washed with 500 ⁇ l of PBS, and then the cells were lysed with 50 ⁇ l of 1x Glo Lysis buffer (Promega #E2661). Of this, 5 ⁇ l was used for luciferase activity measurement, and 5 ⁇ l was used for protein concentration measurement.
- Luciferase activity was determined by detecting the luminescence of coelenterazine h (FUJIFILM #035-22991) to quantify the expression level of Rluc, protein concentration was quantified using the BCA method, and each luciferase activity was calculated as the Rluc/BCA value. bottom.
- a multi-label reader 2030 ARVOTM X (Perkin Elmer) was used for luminescence detection and BCA measurement.
- Western blot HEK293A cells were seeded in a 6-well plate at 5.0 ⁇ 10 5 cells/well and cultured for 24 hours. Thereafter, each compound group was treated by medium exchange, and after 3 hours the cells were washed with 2 ml of PBS and lysed with 200 ⁇ l of RIPA buffer. Lysed cells were disrupted using sonication, centrifuged (20,000 g, 15 min, 4° C.), the supernatant collected and protein concentration quantified. After equal amounts of these lysates were combined, 1 ⁇ SDS sample buffer was added, and the mixture was heat-treated at 95° C. for 3 minutes and used as a sample for SDS-PAGE (5 ⁇ g of protein per lane).
- the membrane was washed three times with TBS-T, and the secondary antibody reaction was performed by adding HRP-labeled secondary antibody (Anti-Rabbit IgG, HRP-Linked Whole Ab Donkey, Cytiva #NA934-1ML) in TBS-T. was performed at room temperature for 3 hours under conditions of 5000-fold dilution. After that, the membrane was washed three times with TBST and detected using Chemi-Lumi One (nacalai #07880-70) and ImageQuant LAS4010.
- HRP-labeled secondary antibody Anti-Rabbit IgG, HRP-Linked Whole Ab Donkey, Cytiva #NA934-1ML
- ELISA method Cells expressing each secretory protein were seeded in a 24-well plate at 1.0 ⁇ 10 4 cells/well and cultured for 24 hours. Thereafter, each compound group was treated by medium exchange under the condition of 1 ml/well, and 120 ⁇ l of medium was collected every 24 hours and stored in a refrigerator. After that, each ELISA kit (mouse IgG, Betyl Lab #E99-131, human proinsulin: Mercodia #10-1118-01, human leptin: Proteintech #KE00095) was used to measure the concentration of the target protein in the medium. A multi-label reader 2030 ARVOTM X (Perkin Elmer) was used for the measurement.
- Test 1 Identification of compounds that enhance the transcriptional activity of the CMV promoter HEK293T cells were transfected with 20 ng per well of a 24-well plate with a plasmid having a luciferase gene inserted downstream of the CMV promoter. The medium was replaced with a medium containing a plant extract. CMV promoter activity was then measured 24 hours later using a luciferase assay. About 1000 types of plant extracts were used in this study. Five types of plant extracts that dramatically enhance the transcriptional activity of the CMV promoter and compounds responsible for the activity (the following eight types) were identified from a proprietary plant (raw drug material) extract library.
- Test 2 Activity of Compounds is Inhibited by Inhibitors of Protein Kinase C (PKC) HEK293A cells constitutively expressing pRL-CMV were treated with Compound X or Compounds #1-#7 at the concentrations indicated in FIG. The cells were cultured in the medium in the presence or absence of PKC inhibitors for 24 hours, and luciferase activity was measured. The results are shown in FIG. Transcriptional activity of the CMV promoter was enhanced in the presence of compounds, and the enhancement was inhibited by PKC inhibitors.
- PKC Protein Kinase C
- HEK293A cells that constitutively express pRL-CMV were cultured in medium supplemented with compound X (100 ng/ml) for 24 hours, and luciferase activity was measured over time. The results are shown in FIG. Enhancement of transcriptional activity of the CMV promoter by Compound X was observed from 1 hour after addition of Compound X.
- Test 3 CMV promoter activity is also enhanced by known PKC activators. was cultured for 24 hours in the presence or absence of a PKC inhibitor in a medium containing the luciferase activity was measured. The results are shown in FIG. CMV promoter transcriptional activity was also enhanced by known PKC activators.
- Compound X, compounds #1-#7 and PKC activators #1-#4 belong to terpenes and are compounds collectively called diterpenes. It is classified into the following three types according to the skeleton.
- Test 4 CMV promoter activity is also enhanced by PKC activators other than diterpenes.
- the cells were cultured in medium containing 6 in the presence or absence of a PKC inhibitor (Ro-318425) for 24 hours, and luciferase activity was measured. The results are shown in FIG. CMV promoter transcriptional activity was also enhanced by PKC activators other than diterpenes.
- Test 5 Compounds also enhance CAG and EF1 promoter activity
- the cells were cultured in media containing PKC activators #1 to #6, and the transcriptional activities of the pCAGGS vector-derived CAG promoter and the pEFBOS vector-derived EF1 promoter were measured. The results are shown in Figures 5-7.
- CAG and EF1 promoter transcriptional activities were enhanced in the presence of compounds or PKC activators, but the enhancement was inhibited by PKC inhibitors. These promoters have binding sites for transcription factors such as SP1, CEBP, AP1, NF- ⁇ B, YY1. Therefore, it is suggested that these compounds and PKC activators activate these promoters through common transcription factors.
- Test 6 Compounds Activate PKC HEK293A cells were treated with Compound X, Compounds #1-#7, PKC activators #1-#6, Compound SC or sodium butyrate ( SB) in the presence or absence of a PKC inhibitor (3 ⁇ M) for 3 hours. Cells were lysed and Western blots were performed using an antibody that specifically recognizes proteins phosphorylated by PKC. The results are shown in FIG. Compound X, compounds #1-#7 and PKC activators #1-#6 activated PKC. PKC activation by Compound SC and sodium butyrate was not confirmed.
- Test 7 Identification of New CMV Promoter Activators HEK293A cells constitutively expressing pRL-CMV were incubated in medium containing compound X and/or compound SC at the concentrations shown in FIG. After culturing for 24 hours in the absence, luciferase activity was measured. The results are shown in FIG. CMV promoter transcriptional activity was also enhanced by compound SC, but this enhancement was not inhibited by PKC inhibitors. Transcriptional activity was further enhanced when compound X and compound SC were used in combination.
- Test 8 Combined use of PKC activator, compound SC and histone deacetylase inhibitor further enhances transcriptional activity of CMV promoter.
- X, compound SC and/or sodium butyrate (SB) were cultured for 24 hours and luciferase activity was measured. The results are shown in FIG. Transcriptional activity of the CMV promoter was highest when compound X, compound SC and sodium butyrate were combined. Similar results were obtained with compounds #1-#7 and PKC activators #1-#6 (FIGS. 11 and 12).
- Test 9 Increased Protein Production by PKC Activator Expression constructs were prepared by inserting cDNAs of mouse IgG antibody heavy chain, mouse IgG antibody light chain, human proinsulin or human leptin downstream of the CMV promoter of a pcDNA vector.
- Mouse IgG antibody and human leptin constructs were transfected into HEK293A cells, respectively.
- the human proinsulin construct was transfected into HEK293A cells to generate HEK293A cells that constitutively express human proinsulin.
- Compound SC or Compound SC plus sodium butyrate (SB) in media containing Compound X, Compounds #1-#7, PKC activators #1-#6 at the concentrations shown in FIGS.
- Test 10 Activation of CMV promoter by genetically engineered PKC activation PKC was genetically engineered to examine the transcriptional activity of the CMV promoter.
- An expression construct was used in which the cDNA for the protein (XXX), the IRES and the cDNA for activated PKC were linked downstream of the CMV promoter.
- the activated PKC the 334-695 amino acid region of human PKC ⁇ (PKC ⁇ -CA) and the constitutively activated form (PKC ⁇ CA-M489V) in which the 489th methionine in the 326-672 amino acid region of human PKC ⁇ was changed to valine were used.
- PKC ⁇ -CA the 334-695 amino acid region of human PKC ⁇
- PDC ⁇ CA-M489V constitutively activated form
- XXX Rluc, mouse IgG antibody (heavy chain, light chain), and human leptin gene were inserted. Schematic representations of these constructs are shown in FIG.
- An expression construct was prepared by inserting Rluc cDNA, IRES, and PKC ⁇ -CA or PKC ⁇ CA-M489V cDNA downstream of the CMV promoter in a pcDNA vector.
- a control construct lacking PKC was also made.
- HEK293A cells were transfected with these constructs at concentrations of 6.25-200 ng/well, cultured for 24 hours, and luciferase activity was measured. The results are shown in FIG.
- the PKC group had higher luciferase activity. This result indicates that the transcriptional activity of the CMV promoter is enhanced by positive feedback. Similar results were obtained with an expression construct using the P2A peptide sequence, a 2A self-cleaving peptide sequence, instead of the IRES (Fig. 21).
- a mouse IgG antibody heavy chain or light chain cDNA, IRES, and PKC ⁇ -CA or PKC ⁇ CA-M489V cDNA were inserted downstream of the CMV promoter in a pcDNA vector to prepare an expression construct.
- a control construct lacking PKC was also made.
- HEK293A cells were transfected with combined IgG antibody heavy and light chain constructs at a concentration of 100 ng/well, cultured for 24 hours, and the concentration of IgG antibodies in the medium was measured by ELISA every 24 hours for 4 days. bottom. The results are shown in FIG. In the PKC group, IgG antibody production was enhanced. This result indicates that genetically engineered PKC activation can activate the CMV promoter and enhance protein production.
- An expression construct was prepared by inserting human leptin cDNA, IRES, and PKC ⁇ CA-M489V cDNA downstream of the CMV promoter of the pcDNA vector.
- a control construct lacking PKC was also made.
- HEK293A cells were transfected with these constructs at a concentration of 100 ng/well, cultured for 24 hours, and the concentration of leptin in the medium was measured by ELISA over 4 days. The results are shown in FIG. In the PKC group, leptin production was enhanced. This result indicates that genetically engineered PKC activation can activate the CMV promoter and enhance protein production.
- Test 11 Search for Target Protein of Compound SC
- compound X was investigated for naphthalenesulfonamide derivatives having the following structure, including compound SC.
- HEK293A cells constitutively expressing pRL-CMV were cultured for 24 hours in the presence or absence of compound X (100 ng/ml) in a medium containing any of the naphthalenesulfonamide derivatives at the concentrations shown in FIG. , luciferase activity was measured. The results are shown in FIG.
- the transcriptional activity of the CMV promoter was not changed by the naphthalenesulfonamide derivative alone, but was enhanced when the naphthalenesulfonamide derivative was used in combination with compound X.
- the naphthalenesulfonamide derivative W-7 has been utilized as a calmodulin inhibitor, and compounds SC and SC-9 also act as calmodulin inhibitors and are thought to exhibit a synergistic effect with compound X.
- Test 12 Transcriptional activity of CMV promoter is enhanced by PKC activator and genetically engineered PML expression.
- ⁇ 10 or PML-Ring) cDNA was inserted to generate an expression construct (Fig. 25). Wild-type PML and PML ⁇ 1-10 constructs were transfected into HEK293A cells, cultured in the presence or absence of compound X for 24 hours, and luciferase activity was measured. The results are shown in FIG. The luciferase activity in the presence of compound X was higher in cells transfected with wild-type PML, PML ⁇ 1 and PML ⁇ 6-10 compared to control ( ⁇ ) cells.
- HEK293A cells were also transfected with constructs containing PML ⁇ 9 or PML-Ring, cultured for 24 hours in the presence or absence of compound X, SB, compound SC, or a combination thereof, and luciferase activity was measured.
- the results are shown in FIG.
- the cells into which PML ⁇ 9 or PML-Ring was introduced had higher luciferase activity under all conditions than the control (Mock) cells. This result suggests that the RING region encompassing PML positions 47-106 also enhances compound X-enhanced transcriptional activity of the CMV promoter.
- the luciferase activity was higher than the control ( ⁇ ) cells even in the absence of compound X.
- Test 13 Genetic Engineering Expression of a PKC Activator and a Peptide Containing the RING Region Enhances the Transcriptional Activity of the CMV Promoter
- the RING region has a common sequence among various proteins. BLAST analysis was performed on the amino acid sequence of the RING region of PML, and proteins shown in FIG. 28 were identified as proteins having similar sequences. Some cysteine and histidine residues were highly conserved in the RING regions of these proteins. Several proteins with different degrees of similarity to the PML RING region were selected and used for the following analysis.
- the cDNA of Rluc, IRES, and the cDNA of the RING region of PML, TRIM3, TRIM56, COPI, Pex10, BRCA1 or HTLF were inserted downstream of the CMV promoter of the pcDNA vector to prepare an expression construct (Fig. 29).
- These expression constructs were transfected into HEK293A cells, cultured in the presence or absence of compound X for 24 hours, and luciferase activity was measured. The results are shown in FIG.
- the luciferase activity in the presence of compound X was higher in the cells introduced with the RING region than in the control (-) cells. This result suggests that the RING region of various proteins further enhances the CMV promoter transcriptional activity enhanced by Compound X.
- the luciferase activity was higher than in the control (-) cells even in the absence of compound X.
- Test 14 Enhancement of transcriptional activity of CMV promoter by PML depends on mRNA expression.
- a cDNA of PML ⁇ 9 introduced with a stop codon or PML ⁇ 9 introduced with a stop codon at position 7) was inserted to prepare an expression construct (FIG. 31, left).
- the cDNA of Rluc and the cDNA of wild-type PML or defective mutant PML (PML ⁇ 9, PML ⁇ 10 or PML-RING) were inserted downstream of the CMV promoter of the pcDNA vector to prepare an expression construct (FIG. 31, right). .
- the PML mRNA is transcribed but not translated into protein.
- constructs were transfected into HEK293A cells, cultured in the presence or absence of Compound X for 24 hours, and luciferase activity was measured. The results are shown in FIG. Both constructs showed higher luciferase activity than the control in the presence and absence of compound X. This result suggests that enhancement of the transcriptional activity of the CMV promoter by PML depends on the expression of the mRNA encoding the PML Pro region or RING region, rather than the PML protein.
- the present disclosure can be used for the production of biopharmaceuticals, as it enables increased production of the protein of interest.
Abstract
The present disclosure provides a method for producing a target protein, said method comprising culturing, under conditions that activate protein kinase C (PKC), cells that contain a nucleic acid that encodes the target protein and is operably linked to a promoter that contains a binding site for at least one transcription factor selected from SP1, CEBP, AP1, NF-κB, and YY1.
Description
本特許出願は、日本国特許出願第2021-144198号について優先権を主張するものであり、ここに参照することによって、その全体が本明細書中へ組み込まれるものとする。
本開示は、タンパク質の製造方法に関するものである。 This patent application claims priority from Japanese Patent Application No. 2021-144198, which is incorporated herein in its entirety by reference.
The present disclosure relates to methods for producing proteins.
本開示は、タンパク質の製造方法に関するものである。 This patent application claims priority from Japanese Patent Application No. 2021-144198, which is incorporated herein in its entirety by reference.
The present disclosure relates to methods for producing proteins.
抗体などの生物製剤(バイオ医薬品)は、これまでに困難であった多くの慢性及び急性疾患の治療を可能とし、現代医療に必要不可欠な医薬品になりつつある。しかし、バイオ医薬品の価格は非常に高額であり、多くの人がその恩恵を享受出来るには至っていない。その最大の原因として、通常、バイオ医薬品の生産には培養細胞が用いられ、その培養容積あたりの産生量が限られていることが挙げられる。
Antibodies and other biological products (biopharmaceuticals) have made it possible to treat many chronic and acute diseases that were previously difficult to treat, and are becoming essential pharmaceuticals for modern medicine. However, the price of biopharmaceuticals is very high, and many people cannot enjoy the benefits. The biggest reason for this is that cultured cells are usually used for the production of biopharmaceuticals, and the amount of production per culture volume is limited.
具体的には、バイオ医薬品は、CMVプロモーターに代表される強力なプロモーターの下流に目的とするタンパク質(抗体、生理活性ペプチドなど)のcDNAをつなぎ、HEKやCHO等の哺乳類培養細胞で発現させることで製造される。バイオ医薬品の生産量は、細胞のタンパク質生産能、培養容積および培養時間により決定されるため、製造コストを抑制しながら生産量を増加させるには、細胞のタンパク質生産能を高める必要がある。
Specifically, biopharmaceuticals are produced by connecting the cDNA of the target protein (antibody, bioactive peptide, etc.) downstream of a strong promoter represented by the CMV promoter, and expressing it in cultured mammalian cells such as HEK and CHO. Manufactured in Since the production of biopharmaceuticals is determined by the protein-producing capacity of cells, the culture volume and the culture time, it is necessary to increase the protein-producing capacity of cells in order to increase production while suppressing production costs.
本開示の目的は、培養細胞を用いるタンパク質の製造において、目的のタンパク質の生産量を高めることである。
The purpose of the present disclosure is to increase the yield of the target protein in protein production using cultured cells.
本発明者らは、PKCを活性化することにより、CMVプロモーターなどのプロモーターの転写活性を高められること、および、PKCを活性化する新たな方法を見出し、それにより培養細胞における目的のタンパク質の生産量を高められることを明らかにした。
The present inventors have found that activating PKC can enhance the transcriptional activity of promoters such as the CMV promoter, and have found a new method for activating PKC, thereby producing the desired protein in cultured cells. It was revealed that the amount could be increased.
従って、ある態様では、本開示は、目的のタンパク質の製造方法であって、SP1、CEBP、AP1、NF-κBおよびYY1から選択される少なくとも1つの転写因子の結合部位を含むプロモーターに作動可能に連結した目的のタンパク質をコードする核酸を含む細胞を、PKCを活性化する条件下で培養することを含み、
PKCを活性化する条件下での培養が、以下のいずれかである、方法を提供する:
(1)PKC活性化剤およびカルモジュリン阻害剤の存在下で前記細胞を培養する、
(2)活性化型PKCを前記細胞に発現させ、カルモジュリン阻害剤の存在下で培養する、
(3)Pro領域またはRING領域を含むペプチドをコードする核酸を前記細胞に発現させ、PKC活性化剤の存在下で培養する、および、
(4)
から選択される化合物またはそのエステル、塩もしくは溶媒和物の存在下で前記細胞を培養する。
Accordingly, in one aspect, the present disclosure provides a method for producing a protein of interest operably linked to a promoter comprising a binding site for at least one transcription factor selected from SP1, CEBP, AP1, NF-κB and YY1. culturing a cell containing a nucleic acid encoding a linked protein of interest under conditions that activate PKC;
A method is provided wherein culturing under conditions that activate PKC is any of the following:
(1) culturing said cells in the presence of a PKC activator and a calmodulin inhibitor;
(2) expressing activated PKC in the cells and culturing in the presence of a calmodulin inhibitor;
(3) expressing a nucleic acid encoding a peptide containing a Pro region or a RING region in said cells and culturing in the presence of a PKC activator;
(4)
The cells are cultured in the presence of a compound selected from or an ester, salt or solvate thereof.
PKCを活性化する条件下での培養が、以下のいずれかである、方法を提供する:
(1)PKC活性化剤およびカルモジュリン阻害剤の存在下で前記細胞を培養する、
(2)活性化型PKCを前記細胞に発現させ、カルモジュリン阻害剤の存在下で培養する、
(3)Pro領域またはRING領域を含むペプチドをコードする核酸を前記細胞に発現させ、PKC活性化剤の存在下で培養する、および、
(4)
A method is provided wherein culturing under conditions that activate PKC is any of the following:
(1) culturing said cells in the presence of a PKC activator and a calmodulin inhibitor;
(2) expressing activated PKC in the cells and culturing in the presence of a calmodulin inhibitor;
(3) expressing a nucleic acid encoding a peptide containing a Pro region or a RING region in said cells and culturing in the presence of a PKC activator;
(4)
ある態様では、本開示は、目的の遺伝子の転写を増強する方法であって、SP1、CEBP、AP1、NF-κBおよびYY1から選択される少なくとも1つの転写因子の結合部位を含むプロモーターに作動可能に連結した当該遺伝子の核酸を含む細胞を、PKCを活性化する条件下で培養することを含み、
PKCを活性化する条件下での培養が、以下のいずれかである、方法を提供する:
(1)PKC活性化剤およびカルモジュリン阻害剤の存在下で前記細胞を培養する、
(2)活性化型PKCを前記細胞に発現させ、カルモジュリン阻害剤の存在下で培養する、
(3)Pro領域またはRING領域を含むペプチドをコードする核酸を前記細胞に発現させ、PKC活性化剤の存在下で培養する、および、
(4)化合物Xおよび化合物#1~#7から選択される化合物またはそのエステル、塩もしくは溶媒和物の存在下で前記細胞を培養する。 In one aspect, the present disclosure provides a method of enhancing transcription of a gene of interest operable on a promoter comprising a binding site for at least one transcription factor selected from SP1, CEBP, AP1, NF-κB and YY1. culturing under conditions that activate PKC a cell containing the nucleic acid of the gene linked to
A method is provided wherein culturing under conditions that activate PKC is any of the following:
(1) culturing said cells in the presence of a PKC activator and a calmodulin inhibitor;
(2) expressing activated PKC in the cells and culturing in the presence of a calmodulin inhibitor;
(3) expressing a nucleic acid encoding a peptide containing a Pro region or a RING region in said cells and culturing in the presence of a PKC activator;
(4) culturing the cells in the presence of compound X and a compound selected fromcompounds # 1 to #7 or an ester, salt or solvate thereof;
PKCを活性化する条件下での培養が、以下のいずれかである、方法を提供する:
(1)PKC活性化剤およびカルモジュリン阻害剤の存在下で前記細胞を培養する、
(2)活性化型PKCを前記細胞に発現させ、カルモジュリン阻害剤の存在下で培養する、
(3)Pro領域またはRING領域を含むペプチドをコードする核酸を前記細胞に発現させ、PKC活性化剤の存在下で培養する、および、
(4)化合物Xおよび化合物#1~#7から選択される化合物またはそのエステル、塩もしくは溶媒和物の存在下で前記細胞を培養する。 In one aspect, the present disclosure provides a method of enhancing transcription of a gene of interest operable on a promoter comprising a binding site for at least one transcription factor selected from SP1, CEBP, AP1, NF-κB and YY1. culturing under conditions that activate PKC a cell containing the nucleic acid of the gene linked to
A method is provided wherein culturing under conditions that activate PKC is any of the following:
(1) culturing said cells in the presence of a PKC activator and a calmodulin inhibitor;
(2) expressing activated PKC in the cells and culturing in the presence of a calmodulin inhibitor;
(3) expressing a nucleic acid encoding a peptide containing a Pro region or a RING region in said cells and culturing in the presence of a PKC activator;
(4) culturing the cells in the presence of compound X and a compound selected from
ある態様では、本願は、目的のタンパク質を製造するためのキットであって、
(1)PKC活性化剤およびカルモジュリン阻害剤、
(2)活性化型PKCをコードする核酸およびカルモジュリン阻害剤、
(3)PKC活性化剤およびPro領域またはRING領域を含むペプチドをコードする核酸、または、
(4)化合物Xおよび化合物#1~#7から選択される化合物またはそのエステル、塩もしくは溶媒和物、
を含み、
目的のタンパク質の製造が、SP1、CEBP、AP1、NF-κBおよびYY1から選択される少なくとも1つの転写因子の結合部位を含むプロモーターに作動可能に連結した目的のタンパク質をコードする核酸を含む細胞を培養することを含む、キットを提供する。 In one aspect, the present application provides a kit for producing a protein of interest, comprising:
(1) PKC activators and calmodulin inhibitors,
(2) a nucleic acid encoding activated PKC and a calmodulin inhibitor;
(3) a nucleic acid encoding a peptide comprising a PKC activator and a Pro region or a RING region, or
(4) a compound selected from compound X andcompounds # 1 to #7 or an ester, salt or solvate thereof;
including
Production of the protein of interest comprises a cell comprising a nucleic acid encoding the protein of interest operably linked to a promoter comprising a binding site for at least one transcription factor selected from SP1, CEBP, AP1, NF-κB and YY1. A kit is provided, comprising culturing.
(1)PKC活性化剤およびカルモジュリン阻害剤、
(2)活性化型PKCをコードする核酸およびカルモジュリン阻害剤、
(3)PKC活性化剤およびPro領域またはRING領域を含むペプチドをコードする核酸、または、
(4)化合物Xおよび化合物#1~#7から選択される化合物またはそのエステル、塩もしくは溶媒和物、
を含み、
目的のタンパク質の製造が、SP1、CEBP、AP1、NF-κBおよびYY1から選択される少なくとも1つの転写因子の結合部位を含むプロモーターに作動可能に連結した目的のタンパク質をコードする核酸を含む細胞を培養することを含む、キットを提供する。 In one aspect, the present application provides a kit for producing a protein of interest, comprising:
(1) PKC activators and calmodulin inhibitors,
(2) a nucleic acid encoding activated PKC and a calmodulin inhibitor;
(3) a nucleic acid encoding a peptide comprising a PKC activator and a Pro region or a RING region, or
(4) a compound selected from compound X and
including
Production of the protein of interest comprises a cell comprising a nucleic acid encoding the protein of interest operably linked to a promoter comprising a binding site for at least one transcription factor selected from SP1, CEBP, AP1, NF-κB and YY1. A kit is provided, comprising culturing.
ある態様では、本願は、作動可能に連結されている、SP1、CEBP、AP1、NF-κBおよびYY1から選択される少なくとも1つの転写因子の結合部位を含むプロモーター、タンパク質をコードする核酸、内部リボソーム進入部位(IRES)または2A自己切断ペプチド(2Aペプチド)配列、および、活性化型PKCをコードする核酸を含む、発現コンストラクトを提供する。
In one aspect, the present application provides a promoter comprising a binding site for at least one transcription factor selected from SP1, CEBP, AP1, NF-κB and YY1, operably linked, a nucleic acid encoding a protein, an internal ribosome Expression constructs are provided that include an entry site (IRES) or 2A self-cleaving peptide (2A peptide) sequence and a nucleic acid encoding activated PKC.
ある態様では、本願は、作動可能に連結されている、SP1、CEBP、AP1、NF-κBおよびYY1から選択される少なくとも1つの転写因子の結合部位を含むプロモーター、タンパク質をコードする核酸、および、Pro領域またはRING領域を含むペプチドをコードする核酸を含む、発現コンストラクトを提供する。
In one aspect, the present application provides, operably linked, a promoter comprising a binding site for at least one transcription factor selected from SP1, CEBP, AP1, NF-κB and YY1, a nucleic acid encoding a protein, and An expression construct is provided that includes a nucleic acid encoding a peptide that includes a Pro region or a RING region.
ある態様では、本願は、化合物Xおよび化合物#1~#7から選択される化合物またはそのエステル、塩もしくは溶媒和物を含む、PKCを活性化するための組成物を提供する。
In one aspect, the present application provides a composition for activating PKC comprising Compound X and a compound selected from Compounds #1-#7 or an ester, salt or solvate thereof.
本開示により、目的のタンパク質の生産量を高めることができる。
The present disclosure can increase the production of the target protein.
特に具体的な定めのない限り、本開示で使用される用語は、有機化学、医学、薬学、分子生物学、微生物学等の分野における当業者に一般に理解されるとおりの意味を有する。以下にいくつかの本開示で使用される用語についての定義を記載するが、これらの定義は、本開示において、一般的な理解に優先する。
Unless otherwise specified, the terms used in this disclosure have the meanings commonly understood by those skilled in the art in the fields of organic chemistry, medicine, pharmacy, molecular biology, microbiology, and the like. Listed below are definitions for some terms used in this disclosure, which definitions take precedence over general understanding in this disclosure.
本開示のタンパク質の製造方法は、目的のタンパク質をコードする核酸を培養細胞に導入し、タンパク質を発現させて回収する、遺伝子工学的手法を利用するものである。かかる遺伝子工学的手法は、当分野で周知であり、文献に記載される方法などに準じて行うことができる(例えば、Molecular Cloning, T. Maniatis et al., CSH Laboratory (1983)、DNA Cloning, DM.Glover, IRL PRESS (1985))。
The method for producing the protein of the present disclosure utilizes genetic engineering techniques in which a nucleic acid encoding a target protein is introduced into cultured cells, and the protein is expressed and recovered. Such genetic engineering techniques are well known in the art and can be carried out according to methods described in literature (for example, Molecular Cloning, T. Maniatis et al., CSH Laboratory (1983), DNA Cloning, D.M. Glover, IRL PRESS (1985)).
本開示において、プロモーターは、SP1、CEBP、AP1、NF-κBおよびYY1から選択される少なくとも1つの転写因子の結合部位を含むものであればよい。プロモーターは、さらに他の転写因子、例えばCREBの結合部位を含んでもよい。ある実施態様では、プロモーターは、SP1、CEBP、AP1、NF-κBおよびYY1の結合部位を含む。ある実施態様では、プロモーターは、SP1、CEBP、AP1、NF-κB、YY1およびCREBの結合部位を含む。使用し得るプロモーターの例として、CMVプロモーター、CAGプロモーターおよびEF1プロモーター、特にCMVプロモーターが挙げられる。これらの転写因子、結合部位、プロモーターは当分野で周知であり、文献に記載される方法などに準じて使用できる。
In the present disclosure, the promoter may contain a binding site for at least one transcription factor selected from SP1, CEBP, AP1, NF-κB and YY1. The promoter may also contain binding sites for other transcription factors, such as CREB. In some embodiments, the promoter comprises binding sites for SP1, CEBP, AP1, NF-κB and YY1. In some embodiments, the promoter comprises binding sites for SP1, CEBP, AP1, NF-κB, YY1 and CREB. Examples of promoters that may be used include the CMV promoter, the CAG promoter and the EF1 promoter, especially the CMV promoter. These transcription factors, binding sites, and promoters are well known in the art and can be used according to methods described in the literature.
本開示において、「作動可能に連結した」とは、プロモーター、IRESおよび2Aペプチド配列などの調節配列エレメントと、タンパク質をコードする核酸が、タンパク質の発現を可能とする様式で連結されていることを意味し、各DNAの3’末端と、その下流のDNAの5’末端とは、直接連結していてもよく、その間に任意のDNA配列が存在してもよい。
In the present disclosure, "operably linked" means that the regulatory sequence elements, such as the promoter, IRES and 2A peptide sequences, and the nucleic acid encoding the protein are linked in a manner that allows expression of the protein. meaning that the 3' end of each DNA and the 5' end of its downstream DNA may be directly linked, and any DNA sequence may exist between them.
目的のタンパク質は、使用する培養細胞に対して許容し得ない毒性がない限り、限定されない。例えば、目的のタンパク質は、免疫グロブリン、レプチン、インスリンまたはこれらの断片などの、バイオ医薬品の有効成分であり得る。目的のタンパク質をコードする核酸は、タンパク質のアミノ酸配列情報およびそれをコードする核酸の配列情報に基づき、例えば、通常のDNA合成やRT-PCRによる増幅などによって、製造することができる。
The target protein is not limited as long as it does not have unacceptable toxicity to the cultured cells used. For example, the protein of interest can be the active ingredient of a biopharmaceutical such as immunoglobulin, leptin, insulin or fragments thereof. A nucleic acid encoding a protein of interest can be produced based on the amino acid sequence information of the protein and the sequence information of the nucleic acid encoding it, for example, by conventional DNA synthesis or amplification by RT-PCR.
プロモーターと目的のタンパク質をコードする核酸を発現ベクターに組み込むことにより、発現コンストラクトを作製することができる。ここで用いる発現ベクターは、用いる宿主や目的等に応じて適宜選択することができ、プラスミド、ファージベクター、ウイルスベクター等が挙げられる。例えば、pKCR、pCDM8、pGL2、pcDNA3.1、pRc/RSV、pRc/CMVなどのプラスミドベクターや、レトロウイルスベクター、アデノウイルスベクター、アデノ関連ウイルスベクターなどのウイルスベクターが挙げられる。ベクターは、選択用マーカー遺伝子、ターミネーターなどの因子を適宜有していてもよい。
An expression construct can be created by incorporating a promoter and a nucleic acid encoding the target protein into an expression vector. The expression vector used here can be appropriately selected according to the host to be used, the purpose, etc. Examples thereof include plasmids, phage vectors, virus vectors, and the like. Examples include plasmid vectors such as pKCR, pCDM8, pGL2, pcDNA3.1, pRc/RSV and pRc/CMV, and viral vectors such as retroviral vectors, adenoviral vectors and adeno-associated viral vectors. The vector may optionally have elements such as a selection marker gene and a terminator.
発現コンストラクトで宿主細胞を形質転換することにより、プロモーターと目的のタンパク質をコードする核酸を含む細胞を作製することができる。宿主細胞は典型的には動物細胞であり、例えば、HEK293A細胞、HEK293T細胞、CHO細胞、COS細胞、Vero細胞、HeLa細胞、L929細胞、BALB/c3T3細胞、C127細胞などが挙げられる。
By transforming a host cell with an expression construct, a cell containing a promoter and a nucleic acid encoding the desired protein can be produced. Host cells are typically animal cells such as HEK293A cells, HEK293T cells, CHO cells, COS cells, Vero cells, HeLa cells, L929 cells, BALB/c3T3 cells, C127 cells and the like.
宿主細胞への発現ベクターの導入方法としては、宿主細胞に適合した通常の導入方法を用いれば良い。具体的にはリン酸カルシウム法、DEAE-デキストラン法、エレクトロポレーション法、リポフェクション法などが挙げられる。かくして得られた形質転換細胞は、目的のタンパク質を一過性に発現してもよく、また、目的のタンパク質を安定に発現する細胞株を樹立してもよい。
As a method for introducing the expression vector into the host cell, a normal introduction method suitable for the host cell may be used. Specific examples include calcium phosphate method, DEAE-dextran method, electroporation method, lipofection method and the like. The transformed cells thus obtained may transiently express the protein of interest, or a cell line stably expressing the protein of interest may be established.
形質転換細胞を、PKCを活性化する条件下で培養することにより、目的のタンパク質を効率よく製造することができる。各細胞の培養に適する培地、培養時間、培養温度等の培養条件は、当業者に周知であり、適宜選択すればよい。例えば、PKCを活性化する条件下で、1時間以上、2時間以上、4時間以上、6時間以上、8時間以上、12時間以上、18時間以上、24時間以上、48時間以上、かつ、60時間以内、48時間以内、36時間以内、30時間以内または24時間以内、細胞を培養した後に、目的のタンパク質を回収する。得られたタンパク質は、一般的な生化学的精製手段により、さらに単離・精製することができる。ここで精製手段としては、塩析、イオン交換クロマトグラフィー、吸着クロマトグラフィー、アフィニティークロマトグラフィー、ゲルろ過クロマトグラフィー等が挙げられる。
By culturing transformed cells under conditions that activate PKC, the target protein can be produced efficiently. Culture conditions such as medium, culture time, and culture temperature suitable for culturing each cell are well known to those skilled in the art, and may be appropriately selected. For example, under conditions that activate PKC, 1 hour or more, 2 hours or more, 4 hours or more, 6 hours or more, 8 hours or more, 12 hours or more, 18 hours or more, 24 hours or more, 48 hours or more, and 60 The protein of interest is recovered within hours, within 48 hours, within 36 hours, within 30 hours or within 24 hours after culturing the cells. The resulting protein can be further isolated and purified by common biochemical purification means. Examples of purification means include salting out, ion exchange chromatography, adsorption chromatography, affinity chromatography, gel filtration chromatography and the like.
ある実施態様では、PKCを活性化する条件は、(1)PKC活性化剤およびカルモジュリン阻害剤の存在下で前記細胞を培養することである。
In one embodiment, the conditions for activating PKC are (1) culturing the cells in the presence of a PKC activator and a calmodulin inhibitor.
PKCは、基質タンパク質のセリン残基およびスレオニン残基のヒドロキシル基をリン酸化するタンパク質キナーゼの一種であり、10種類以上のアイソザイムが知られている。アイソザイムはその構造、活性化の機序、生理活性によって在来型(α、βI、βII、γ)、新型(δ、ε、θ、η)、非典型(ζ、Mζ、ι/λ)の3種類のサブファミリーに分類される。本開示において、PKCは、好ましくは在来型PKCアイソザイムまたは新型PKCアイソザイム、特に好ましくはPKCαまたはPKCδである。
PKC is a type of protein kinase that phosphorylates the hydroxyl groups of serine and threonine residues of substrate proteins, and more than 10 isoenzymes are known. Isozymes are classified into conventional (α, βI, βII, γ), novel (δ, ε, θ, η) and atypical (ζ, Mζ, ι/λ) types depending on their structures, activation mechanisms, and physiological activities. It is classified into three subfamilies. In the present disclosure, PKC is preferably a conventional PKC isozyme or a new PKC isozyme, particularly preferably PKCα or PKCδ.
本開示において、「PKC活性化剤」は、PKCのキナーゼ活性を増強する物質を意味する。2種以上のPKC活性化剤を併用してもよい。
In the present disclosure, "PKC activator" means a substance that enhances the kinase activity of PKC. Two or more PKC activators may be used in combination.
ある実施態様では、PKC活性化剤として、式(I):
{式中、
R1は、H、ハロゲン、-OH、C1-6アルキル、C2-6アルケニル、C2-6アルキニル、C1-6アルコキシ、C6-14アリールまたは-OC(O)R3であり、ここで、C1-6アルキル、C2-6アルケニル、C2-6アルキニル、C1-6アルコキシまたはアリールは、同一または異なる、1個~3個のハロゲンにより置換されていてもよく、
R2は、C6-12アルキル、C6-12アルケニル、C6-12アルキニルまたはC6-12アルコキシであり、ここで、C6-12アルキル、C6-12アルケニル、C6-12アルキニルまたはC6-12アルコキシは、同一または異なる、1個~3個のハロゲンにより置換されていてもよく、
R3は、C1-6アルキル、C2-6アルケニル、C2-6アルキニル、アミノまたはC6-14アリールであり、ここでC1-6アルキル、C2-6アルケニル、C2-6アルキニルまたはC6-14アリールは、同一または異なる、1個~3個のハロゲンにより置換されていてもよい}、
式(II):
{式中、
R4は、H、ハロゲン、-OH、C1-18アルキル、C2-18アルケニル、C2-18アルキニル、C1-18アルコキシまたは-OC(O)R6であり、ここで、C1-18アルキル、C2-18アルケニル、C2-18アルキニルまたはC1-18アルコキシは、同一または異なる、1個~3個のハロゲンにより置換されていてもよく、
R5は、C1-6アルキル、C2-6アルケニル、C2-6アルキニルまたはアミノであり、ここで、C1-6アルキル、C2-6アルケニルまたはC2-6アルキニルは、同一または異なる、1個~3個のハロゲンにより置換されていてもよく、
R6は、C1-18アルキル、C2-18アルケニル、C2-18アルキニルまたはアミノであり、ここでC1-18アルキル、C2-18アルケニルまたはC2-18アルキニルは、同一または異なる、1個~3個のハロゲンにより置換されていてもよい}、
または、
式(III):
{式中、
R7は、H、C1-18アルキル、C2-18アルケニル、C2-18アルキニルまたは-C(O)R9であり、ここで、C1-18アルキル、C2-18アルケニルまたはC2-18アルキニルは、同一または異なる、1個~3個のハロゲンにより置換されていてもよく、
R8は、H、C1-18アルキル、C2-18アルケニル、C2-18アルキニルまたは-C(O)R9であり、ここで、C1-18アルキル、C2-18アルケニルまたはC2-18アルキニルは、同一または異なる、1個~3個のハロゲンにより置換されていてもよく、
R9は、C1-18アルキル、C2-18アルケニル、C2-18アルキニルまたはアミノであり、ここでC1-18アルキル、C2-18アルケニルまたはC2-18アルキニルは、同一または異なる、1個~3個のハロゲンにより置換されていてもよい}
の化合物またはそのエステル、塩もしくは溶媒和物を使用し得る。 In one embodiment, the PKC activator is of formula (I):
{In the formula,
R 1 is H, halogen, —OH, C 1-6 alkyl, C 2-6 alkenyl, C 2-6 alkynyl, C 1-6 alkoxy, C 6-14 aryl or —OC(O)R 3 , wherein C 1-6 alkyl, C 2-6 alkenyl, C 2-6 alkynyl, C 1-6 alkoxy or aryl are optionally substituted by 1 to 3 halogens, the same or different,
R 2 is C 6-12 alkyl, C 6-12 alkenyl, C 6-12 alkynyl or C 6-12 alkoxy, where C 6-12 alkyl, C 6-12 alkenyl, C 6-12 alkynyl or C 6-12 alkoxy is optionally substituted by 1 to 3 halogens, which may be the same or different;
R 3 is C 1-6 alkyl, C 2-6 alkenyl, C 2-6 alkynyl, amino or C 6-14 aryl where C 1-6 alkyl, C 2-6 alkenyl, C 2-6 alkynyl or C 6-14 aryl optionally substituted by 1 to 3 halogens, the same or different},
Formula (II):
{In the formula,
R 4 is H, halogen, —OH, C 1-18 alkyl, C 2-18 alkenyl, C 2-18 alkynyl, C 1-18 alkoxy or —OC(O)R 6 where C 1 -18 alkyl, C 2-18 alkenyl, C 2-18 alkynyl or C 1-18 alkoxy is optionally substituted by 1 to 3 halogens, which may be the same or different;
R 5 is C 1-6 alkyl, C 2-6 alkenyl, C 2-6 alkynyl or amino, wherein C 1-6 alkyl, C 2-6 alkenyl or C 2-6 alkynyl are the same or optionally substituted by 1 to 3 different halogens,
R 6 is C 1-18 alkyl, C 2-18 alkenyl, C 2-18 alkynyl or amino, wherein the C 1-18 alkyl, C 2-18 alkenyl or C 2-18 alkynyl are the same or different , optionally substituted by 1 to 3 halogens},
or,
Formula (III):
{In the formula,
R 7 is H, C 1-18 alkyl, C 2-18 alkenyl, C 2-18 alkynyl or —C(O)R 9 where C 1-18 alkyl, C 2-18 alkenyl or C 2-18 alkynyl is optionally substituted by 1 to 3 halogens, which may be the same or different;
R 8 is H, C 1-18 alkyl, C 2-18 alkenyl, C 2-18 alkynyl or —C(O)R 9 where C 1-18 alkyl, C 2-18 alkenyl or C 2-18 alkynyl is optionally substituted by 1 to 3 halogens, which may be the same or different;
R 9 is C 1-18 alkyl, C 2-18 alkenyl, C 2-18 alkynyl or amino, wherein the C 1-18 alkyl, C 2-18 alkenyl or C 2-18 alkynyl are the same or different , optionally substituted by 1 to 3 halogens}
or an ester, salt or solvate thereof may be used.
R1は、H、ハロゲン、-OH、C1-6アルキル、C2-6アルケニル、C2-6アルキニル、C1-6アルコキシ、C6-14アリールまたは-OC(O)R3であり、ここで、C1-6アルキル、C2-6アルケニル、C2-6アルキニル、C1-6アルコキシまたはアリールは、同一または異なる、1個~3個のハロゲンにより置換されていてもよく、
R2は、C6-12アルキル、C6-12アルケニル、C6-12アルキニルまたはC6-12アルコキシであり、ここで、C6-12アルキル、C6-12アルケニル、C6-12アルキニルまたはC6-12アルコキシは、同一または異なる、1個~3個のハロゲンにより置換されていてもよく、
R3は、C1-6アルキル、C2-6アルケニル、C2-6アルキニル、アミノまたはC6-14アリールであり、ここでC1-6アルキル、C2-6アルケニル、C2-6アルキニルまたはC6-14アリールは、同一または異なる、1個~3個のハロゲンにより置換されていてもよい}、
式(II):
R4は、H、ハロゲン、-OH、C1-18アルキル、C2-18アルケニル、C2-18アルキニル、C1-18アルコキシまたは-OC(O)R6であり、ここで、C1-18アルキル、C2-18アルケニル、C2-18アルキニルまたはC1-18アルコキシは、同一または異なる、1個~3個のハロゲンにより置換されていてもよく、
R5は、C1-6アルキル、C2-6アルケニル、C2-6アルキニルまたはアミノであり、ここで、C1-6アルキル、C2-6アルケニルまたはC2-6アルキニルは、同一または異なる、1個~3個のハロゲンにより置換されていてもよく、
R6は、C1-18アルキル、C2-18アルケニル、C2-18アルキニルまたはアミノであり、ここでC1-18アルキル、C2-18アルケニルまたはC2-18アルキニルは、同一または異なる、1個~3個のハロゲンにより置換されていてもよい}、
または、
式(III):
R7は、H、C1-18アルキル、C2-18アルケニル、C2-18アルキニルまたは-C(O)R9であり、ここで、C1-18アルキル、C2-18アルケニルまたはC2-18アルキニルは、同一または異なる、1個~3個のハロゲンにより置換されていてもよく、
R8は、H、C1-18アルキル、C2-18アルケニル、C2-18アルキニルまたは-C(O)R9であり、ここで、C1-18アルキル、C2-18アルケニルまたはC2-18アルキニルは、同一または異なる、1個~3個のハロゲンにより置換されていてもよく、
R9は、C1-18アルキル、C2-18アルケニル、C2-18アルキニルまたはアミノであり、ここでC1-18アルキル、C2-18アルケニルまたはC2-18アルキニルは、同一または異なる、1個~3個のハロゲンにより置換されていてもよい}
の化合物またはそのエステル、塩もしくは溶媒和物を使用し得る。 In one embodiment, the PKC activator is of formula (I):
R 1 is H, halogen, —OH, C 1-6 alkyl, C 2-6 alkenyl, C 2-6 alkynyl, C 1-6 alkoxy, C 6-14 aryl or —OC(O)R 3 , wherein C 1-6 alkyl, C 2-6 alkenyl, C 2-6 alkynyl, C 1-6 alkoxy or aryl are optionally substituted by 1 to 3 halogens, the same or different,
R 2 is C 6-12 alkyl, C 6-12 alkenyl, C 6-12 alkynyl or C 6-12 alkoxy, where C 6-12 alkyl, C 6-12 alkenyl, C 6-12 alkynyl or C 6-12 alkoxy is optionally substituted by 1 to 3 halogens, which may be the same or different;
R 3 is C 1-6 alkyl, C 2-6 alkenyl, C 2-6 alkynyl, amino or C 6-14 aryl where C 1-6 alkyl, C 2-6 alkenyl, C 2-6 alkynyl or C 6-14 aryl optionally substituted by 1 to 3 halogens, the same or different},
Formula (II):
R 4 is H, halogen, —OH, C 1-18 alkyl, C 2-18 alkenyl, C 2-18 alkynyl, C 1-18 alkoxy or —OC(O)R 6 where C 1 -18 alkyl, C 2-18 alkenyl, C 2-18 alkynyl or C 1-18 alkoxy is optionally substituted by 1 to 3 halogens, which may be the same or different;
R 5 is C 1-6 alkyl, C 2-6 alkenyl, C 2-6 alkynyl or amino, wherein C 1-6 alkyl, C 2-6 alkenyl or C 2-6 alkynyl are the same or optionally substituted by 1 to 3 different halogens,
R 6 is C 1-18 alkyl, C 2-18 alkenyl, C 2-18 alkynyl or amino, wherein the C 1-18 alkyl, C 2-18 alkenyl or C 2-18 alkynyl are the same or different , optionally substituted by 1 to 3 halogens},
or,
Formula (III):
R 7 is H, C 1-18 alkyl, C 2-18 alkenyl, C 2-18 alkynyl or —C(O)R 9 where C 1-18 alkyl, C 2-18 alkenyl or C 2-18 alkynyl is optionally substituted by 1 to 3 halogens, which may be the same or different;
R 8 is H, C 1-18 alkyl, C 2-18 alkenyl, C 2-18 alkynyl or —C(O)R 9 where C 1-18 alkyl, C 2-18 alkenyl or C 2-18 alkynyl is optionally substituted by 1 to 3 halogens, which may be the same or different;
R 9 is C 1-18 alkyl, C 2-18 alkenyl, C 2-18 alkynyl or amino, wherein the C 1-18 alkyl, C 2-18 alkenyl or C 2-18 alkynyl are the same or different , optionally substituted by 1 to 3 halogens}
or an ester, salt or solvate thereof may be used.
本開示において、用語「ハロゲン」は、フッ素、塩素、臭素およびヨウ素から選ばれる原子を意味する。
本開示において、用語「アルキル」は、飽和した、直鎖または分岐鎖の炭化水素基を示す。
本開示において、用語「アルケニル」は、1つまたはそれ以上の二重結合を含む直鎖または分岐鎖の炭化水素を意味する。
本開示において、用語「アルキニル」は、1つまたはそれ以上の三重結合を含む直鎖または分岐鎖の炭化水素を意味する。
本開示において、用語「アルコキシ」は、-O-アルキル(ここで、アルキルは本開示に定義されている通りである)を意味する。 In the present disclosure the term "halogen" means an atom selected from fluorine, chlorine, bromine and iodine.
In this disclosure, the term "alkyl" denotes a saturated, straight or branched chain hydrocarbon group.
In this disclosure, the term "alkenyl" means a straight or branched chain hydrocarbon containing one or more double bonds.
In this disclosure, the term "alkynyl" means a straight or branched chain hydrocarbon containing one or more triple bonds.
In this disclosure, the term "alkoxy" means -O-alkyl, where alkyl is defined in this disclosure.
本開示において、用語「アルキル」は、飽和した、直鎖または分岐鎖の炭化水素基を示す。
本開示において、用語「アルケニル」は、1つまたはそれ以上の二重結合を含む直鎖または分岐鎖の炭化水素を意味する。
本開示において、用語「アルキニル」は、1つまたはそれ以上の三重結合を含む直鎖または分岐鎖の炭化水素を意味する。
本開示において、用語「アルコキシ」は、-O-アルキル(ここで、アルキルは本開示に定義されている通りである)を意味する。 In the present disclosure the term "halogen" means an atom selected from fluorine, chlorine, bromine and iodine.
In this disclosure, the term "alkyl" denotes a saturated, straight or branched chain hydrocarbon group.
In this disclosure, the term "alkenyl" means a straight or branched chain hydrocarbon containing one or more double bonds.
In this disclosure, the term "alkynyl" means a straight or branched chain hydrocarbon containing one or more triple bonds.
In this disclosure, the term "alkoxy" means -O-alkyl, where alkyl is defined in this disclosure.
本開示において、用語「アリール」は、1個の環(例えばフェニル)または複数の縮合環(例えばナフチルまたはアントリル)を有する6~14個の炭素原子の1価芳香族性炭素環式基を意味する。アリールは典型的には、フェニルおよびナフチルを含む。
本開示において、用語「アミノ」は-NH2の基を意味する。 In this disclosure, the term "aryl" means a monovalent aromatic carbocyclic group of 6 to 14 carbon atoms having a single ring (eg phenyl) or multiple condensed rings (eg naphthyl or anthryl). do. Aryl typically includes phenyl and naphthyl.
In this disclosure, the term "amino" means the group -NH2 .
本開示において、用語「アミノ」は-NH2の基を意味する。 In this disclosure, the term "aryl" means a monovalent aromatic carbocyclic group of 6 to 14 carbon atoms having a single ring (eg phenyl) or multiple condensed rings (eg naphthyl or anthryl). do. Aryl typically includes phenyl and naphthyl.
In this disclosure, the term "amino" means the group -NH2 .
本開示において、「エステル」は、インビボまたはインビトロで加水分解され得るエステルを意味し、容易に分解されて親化合物またはその塩を放出するものを含む。好適なエステル基は、例えば、脂肪族カルボン酸、特にアルカン酸、アルケン酸、シクロアルカン酸およびアルカン二酸に由来するもの(ここで、各アルキルまたはアルケニル基は、例えば6個以下の炭素原子を有する)を含む。具体的なエステルの例には、ギ酸エステル、酢酸エステル、プロピオン酸エステル、ブチル酸エステル、アクリル酸エステルおよびエチルコハク酸エステルが含まれる。
In the present disclosure, "ester" means an ester that can be hydrolyzed in vivo or in vitro, including those that are readily degraded to release the parent compound or a salt thereof. Suitable ester groups are, for example, those derived from aliphatic carboxylic acids, especially alkanoic acids, alkenoic acids, cycloalkanoic acids and alkanedioic acids, wherein each alkyl or alkenyl group has, for example, up to 6 carbon atoms. have). Examples of specific esters include formates, acetates, propionates, butyrates, acrylates and ethylsuccinates.
本開示において、「塩」は、化合物の無機または有機酸との塩であり得る。好ましい塩は、無機酸、例えば、塩酸、臭化水素酸、リン酸または硫酸との塩、または、有機カルボン酸またはスルホン酸、例えば、酢酸、トリフルオロ酢酸、プロピオン酸、マレイン酸、フマル酸、リンゴ酸、クエン酸、酒石酸、乳酸、安息香酸またはメタンスルホン酸、エタンスルホン酸、ベンゼンスルホン酸、トルエンスルホン酸、ナフタレンスルホン酸またはナフタレンジスルホン酸との塩である。
In the present disclosure, a "salt" may be a salt of a compound with an inorganic or organic acid. Preferred salts are those with inorganic acids such as hydrochloric, hydrobromic, phosphoric or sulfuric acids, or with organic carboxylic or sulfonic acids such as acetic acid, trifluoroacetic acid, propionic acid, maleic acid, fumaric acid, It is a salt with malic acid, citric acid, tartaric acid, lactic acid, benzoic acid or methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, toluenesulfonic acid, naphthalenesulfonic acid or naphthalenedisulfonic acid.
また、塩は、常套の塩基との塩、例えばアルカリ金属塩(例えばナトリウムまたはカリウム塩)、アルカリ土類金属塩(例えばカルシウムまたはマグネシウム塩)、または、アンモニアまたは有機アミン(例えば、ジエチルアミン、トリエチルアミン、エチルジイソプロピルアミン、プロカイン、ジベンジルアミン、N-メチルモルホリン、ジヒドロアビエチルアミン、メチルピペリジン、L-アルギニン、クレアチン、コリン、L-リジン、エチレンジアミン、ベンザチン、エタノールアミン、メグルミンまたはトロメタミン)から誘導されるアンモニウム塩、特にナトリウム塩であり得る。
Salts can also be salts with customary bases, such as alkali metal salts (for example sodium or potassium salts), alkaline earth metal salts (for example calcium or magnesium salts), or ammonia or organic amines (for example diethylamine, triethylamine, ammonium derived from ethyldiisopropylamine, procaine, dibenzylamine, N-methylmorpholine, dihydroabiethylamine, methylpiperidine, L-arginine, creatine, choline, L-lysine, ethylenediamine, benzathine, ethanolamine, meglumine or tromethamine) It may be a salt, especially the sodium salt.
本開示において、「溶媒和物」は、固体または液体状態で溶媒分子との配位により錯体を形成している化合物を意味する。好適な溶媒和物は水和物である。
In the present disclosure, "solvate" means a compound that forms a complex through coordination with solvent molecules in a solid or liquid state. Preferred solvates are hydrates.
ある実施態様では、式(I)中、
R1は、Hまたは-OC(O)R3であり、
R2は、C6-12アルキルまたはC6-12アルケニルであり、
R3は、C1-6アルキルまたはC6-14アリールである。 In certain embodiments, in Formula (I):
R 1 is H or —OC(O)R 3 ;
R 2 is C 6-12 alkyl or C 6-12 alkenyl,
R 3 is C 1-6 alkyl or C 6-14 aryl.
R1は、Hまたは-OC(O)R3であり、
R2は、C6-12アルキルまたはC6-12アルケニルであり、
R3は、C1-6アルキルまたはC6-14アリールである。 In certain embodiments, in Formula (I):
R 1 is H or —OC(O)R 3 ;
R 2 is C 6-12 alkyl or C 6-12 alkenyl,
R 3 is C 1-6 alkyl or C 6-14 aryl.
ある実施態様では、式(I)中、
R1は、Hまたは-OC(O)R3であり、
R2は、ノニルまたは1,3-ノナジエニルであり、
R3は、メチルまたはフェニルである。 In certain embodiments, in Formula (I):
R 1 is H or —OC(O)R 3 ;
R 2 is nonyl or 1,3-nonadienyl,
R3 is methyl or phenyl.
R1は、Hまたは-OC(O)R3であり、
R2は、ノニルまたは1,3-ノナジエニルであり、
R3は、メチルまたはフェニルである。 In certain embodiments, in Formula (I):
R 1 is H or —OC(O)R 3 ;
R 2 is nonyl or 1,3-nonadienyl,
R3 is methyl or phenyl.
ある実施態様では、式(I)の化合物は、後述する化合物X、化合物#1または化合物#2である。
In some embodiments, the compound of formula (I) is compound X, compound # 1 or compound # 2, described below.
ある実施態様では、式(II)中、
R4は、Hまたは-OC(O)R6であり、
R5は、C1-6アルキルまたはC2-6アルケニルであり、
R6は、C1-18アルキルである。 In certain embodiments, in Formula (II):
R 4 is H or —OC(O)R 6 ;
R 5 is C 1-6 alkyl or C 2-6 alkenyl;
R 6 is C 1-18 alkyl.
R4は、Hまたは-OC(O)R6であり、
R5は、C1-6アルキルまたはC2-6アルケニルであり、
R6は、C1-18アルキルである。 In certain embodiments, in Formula (II):
R 4 is H or —OC(O)R 6 ;
R 5 is C 1-6 alkyl or C 2-6 alkenyl;
R 6 is C 1-18 alkyl.
ある実施態様では、式(II)中、
R4は、Hまたは-OC(O)R6であり、
R5は、メチル、プロピル、sec-ブチルまたはブテニルであり、
R6は、プロピル、ノニルまたはトリデシルである。 In certain embodiments, in Formula (II):
R 4 is H or —OC(O)R 6 ;
R5 is methyl, propyl, sec-butyl or butenyl;
R6 is propyl, nonyl or tridecyl.
R4は、Hまたは-OC(O)R6であり、
R5は、メチル、プロピル、sec-ブチルまたはブテニルであり、
R6は、プロピル、ノニルまたはトリデシルである。 In certain embodiments, in Formula (II):
R 4 is H or —OC(O)R 6 ;
R5 is methyl, propyl, sec-butyl or butenyl;
R6 is propyl, nonyl or tridecyl.
ある実施態様では、式(II)の化合物は、後述する化合物#3、化合物#4、化合物#5、TPA、ホルボール12,13-ジブチラートまたはプロストラチン、特に化合物#3、化合物#4または化合物#5である。
In certain embodiments, the compound of formula (II) is Compound # 3, Compound # 4, Compound # 5, TPA, phorbol 12,13-dibutyrate or prostratin, particularly Compound # 3, Compound # 4 or Compound # 5.
ある実施態様では、式(III)中、
R7は、Hまたは-C(O)R9であり、
R8は、Hまたは-C(O)R9であり、
R9は、C1-18アルキルまたはC2-18アルケニルである。 In certain embodiments, in Formula (III):
R 7 is H or —C(O)R 9 ;
R 8 is H or —C(O)R 9 ;
R 9 is C 1-18 alkyl or C 2-18 alkenyl.
R7は、Hまたは-C(O)R9であり、
R8は、Hまたは-C(O)R9であり、
R9は、C1-18アルキルまたはC2-18アルケニルである。 In certain embodiments, in Formula (III):
R 7 is H or —C(O)R 9 ;
R 8 is H or —C(O)R 9 ;
R 9 is C 1-18 alkyl or C 2-18 alkenyl.
ある実施態様では、式(III)中、
R7は、Hまたは-C(O)R9であり、
R8は、Hまたは-C(O)R9であり、
R9は、ペンタデシルまたはブテニルである。 In certain embodiments, in Formula (III):
R 7 is H or —C(O)R 9 ;
R 8 is H or —C(O)R 9 ;
R9 is pentadecyl or butenyl.
R7は、Hまたは-C(O)R9であり、
R8は、Hまたは-C(O)R9であり、
R9は、ペンタデシルまたはブテニルである。 In certain embodiments, in Formula (III):
R 7 is H or —C(O)R 9 ;
R 8 is H or —C(O)R 9 ;
R9 is pentadecyl or butenyl.
ある実施態様では、式(III)の化合物は、後述する化合物#6、化合物#7またはインゲノール3-アンゲラート、特に化合物#6または化合物#7である。
In one embodiment, the compound of formula (III) is compound # 6, compound # 7 or ingenol 3-angelate, particularly compound # 6 or compound # 7, described below.
式(I)~(III)の化合物またはそのエステル、塩もしくは溶媒和物は、例えば、0.1~10μg/ml、0.1~1μg/ml、0.1~1000ng/ml、2~500ng/mlまたは5~200ng/mlの濃度で培養培地に添加し得る。
Compounds of formulas (I)-(III) or their esters, salts or solvates are, for example, 0.1-10 μg/ml, 0.1-1 μg/ml, 0.1-1000 ng/ml, 2-500 ng /ml or 5-200 ng/ml to the culture medium.
ある実施態様では、PKC活性化剤は、式(I)の化合物またはそのエステル、塩もしくは溶媒和物である。これらは、例えば、0.1~10μg/ml、0.1~1μg/ml、0.1~1000ng/ml、20~500ng/mlまたは50~200ng/mlの濃度で培養培地に添加し得る。
In one embodiment, the PKC activator is a compound of formula (I) or an ester, salt or solvate thereof. These can be added to the culture medium in concentrations of, for example, 0.1-10 μg/ml, 0.1-1 μg/ml, 0.1-1000 ng/ml, 20-500 ng/ml or 50-200 ng/ml.
ある実施態様では、PKC活性化剤は、下記の化合物Xおよび化合物#1~#7から選択される化合物またはそのエステル、塩もしくは溶媒和物である。
In one embodiment, the PKC activator is a compound selected from Compound X and Compounds #1-#7 below or an ester, salt or solvate thereof.
ある実施態様では、PKC活性化剤は化合物Xまたはそのエステル、塩もしくは溶媒和物である。
In some embodiments, the PKC activator is Compound X or an ester, salt or solvate thereof.
公知のPKC活性化剤を使用してもよい。公知のPKC活性化剤として、12-O-テトラデカノイルホルボール13-アセタート(TPA、ホルボール12-ミリスタート13-アセタート(PMA)とも呼ばれる)、プロストラチン、ブリオスタチン1、ブリオスタチン2、FR236924、(-)-インドラクタムV、PEP005、ホルボール12,13-ジブチラート、1-オレオイル-2-アセチル-sn-グリセロール、1-O-ヘキサデシル-2-O-アラキドニル-sn-グリセロール、1,2-ジオクタノイル-sn-グリセロール、PIP2、レシニフェラトキシン、ホルボール12,13-ジヘキサノアート、メゼレイン、インゲノール3-アンゲラート、RHC-80267、DCP-LA、リポキシンA4、(2S,5S)-(E,E)-8-(5-(4-(トリフルオロメチル)フェニル)-2,4-ペンタジエノイルアミノ)ベンゾラクタムなどが例示されるが、これらに限定されない。ある実施態様では、PKC活性化剤は、TPA、プロストラチン、(-)-インドラクタムV、ホルボール12,13-ジブチラート、インゲノール3-アンゲラート、または、(2S,5S)-(E,E)-8-(5-(4-(トリフルオロメチル)フェニル)-2,4-ペンタジエノイルアミノ)ベンゾラクタムである。公知のPKC活性化剤は、当分野で知られている方法、例えば製造業者の推奨する方法により、適宜使用し得る。
A known PKC activator may be used. Known PKC activators include 12-O-tetradecanoylphorbol 13-acetate (TPA, also called phorbol 12-myristate 13-acetate (PMA)), prostratin, bryostatin 1, bryostatin 2, FR236924 , (−)-indolactam V, PEP005, phorbol 12,13-dibutyrate, 1-oleoyl-2-acetyl-sn-glycerol, 1-O-hexadecyl-2-O-arachidonyl-sn-glycerol, 1,2 -dioctanoyl-sn-glycerol, PIP2, resiniferatoxin, phorbol 12,13-dihexanoate, mezerein, ingenol 3-angelate, RHC-80267, DCP-LA, lipoxin A4, (2S,5S)-(E ,E)-8-(5-(4-(trifluoromethyl)phenyl)-2,4-pentadienoylamino)benzolactam and the like, but are not limited thereto. In some embodiments, the PKC activator is TPA, prostratin, (−)-indolactam V, phorbol 12,13-dibutyrate, ingenol 3-angelate, or (2S,5S)-(E,E)- 8-(5-(4-(trifluoromethyl)phenyl)-2,4-pentadienoylamino)benzolactam. Known PKC activators can be used as appropriate by methods known in the art, such as methods recommended by manufacturers.
ある実施態様では、PKC活性化剤は、化合物X、化合物#1~#7、TPA、プロストラチン、(-)-インドラクタムV、ホルボール12,13-ジブチラート、インゲノール3-アンゲラートおよび(2S,5S)-(E,E)-8-(5-(4-(トリフルオロメチル)フェニル)-2,4-ペンタジエノイルアミノ)ベンゾラクタムから選択される化合物またはそのエステル、塩もしくは溶媒和物である。
In certain embodiments, the PKC activator is Compound X, Compounds #1-#7, TPA, prostratin, (−)-indolactam V, phorbol 12,13-dibutyrate, ingenol 3-angelate and (2S,5S )-(E,E)-8-(5-(4-(trifluoromethyl)phenyl)-2,4-pentadienoylamino)benzolactam or an ester, salt or solvate thereof; be.
カルモジュリンは、カルシウムセンサーとして機能する酸性タンパク質であり、細胞内カルシウムレベルを調節する。カルモジュリン阻害剤として、式(IV)
{式中、
nは1~8の整数であり、
Rは、C1-6アルキル、C2-6アルケニル、C2-6アルキニル、C1-6アルコキシ、C6-14アリール、アミノ、ヒドロキシ、COOHまたはCOOR’であり、ここでR’はC1-6アルキルである}
の化合物またはそのエステル、塩もしくは溶媒和物を使用し得る。 Calmodulin is an acidic protein that functions as a calcium sensor, regulating intracellular calcium levels. As a calmodulin inhibitor, formula (IV)
{In the formula,
n is an integer from 1 to 8,
R is C 1-6 alkyl, C 2-6 alkenyl, C 2-6 alkynyl, C 1-6 alkoxy, C 6-14 aryl, amino, hydroxy, COOH or COOR', where R' is C 1-6 alkyl}
or an ester, salt or solvate thereof may be used.
nは1~8の整数であり、
Rは、C1-6アルキル、C2-6アルケニル、C2-6アルキニル、C1-6アルコキシ、C6-14アリール、アミノ、ヒドロキシ、COOHまたはCOOR’であり、ここでR’はC1-6アルキルである}
の化合物またはそのエステル、塩もしくは溶媒和物を使用し得る。 Calmodulin is an acidic protein that functions as a calcium sensor, regulating intracellular calcium levels. As a calmodulin inhibitor, formula (IV)
n is an integer from 1 to 8,
R is C 1-6 alkyl, C 2-6 alkenyl, C 2-6 alkynyl, C 1-6 alkoxy, C 6-14 aryl, amino, hydroxy, COOH or COOR', where R' is C 1-6 alkyl}
or an ester, salt or solvate thereof may be used.
ある実施態様では、式(IV)中、
nは4~6の整数であり、
Rは、C1-6アルキル、C6-14アリールまたはアミノである。 In certain embodiments, in Formula (IV):
n is an integer from 4 to 6,
R is C 1-6 alkyl, C 6-14 aryl or amino.
nは4~6の整数であり、
Rは、C1-6アルキル、C6-14アリールまたはアミノである。 In certain embodiments, in Formula (IV):
n is an integer from 4 to 6,
R is C 1-6 alkyl, C 6-14 aryl or amino.
ある実施態様では、式(IV)中、
nは4または6の整数であり、
Rは、メチル、フェニルまたはアミノである。 In certain embodiments, in Formula (IV):
n is an integer of 4 or 6;
R is methyl, phenyl or amino.
nは4または6の整数であり、
Rは、メチル、フェニルまたはアミノである。 In certain embodiments, in Formula (IV):
n is an integer of 4 or 6;
R is methyl, phenyl or amino.
式(IV)の化合物には、例えば、SC-9、SC-10、W-7が含まれる。式(IV)の化合物は、例えば、0.5~100μg/ml、1~50μg/mlまたは2~10μg/mlの濃度で使用し得る。
The compounds of formula (IV) include, for example, SC-9, SC-10 and W-7. Compounds of formula (IV) may be used at concentrations of, for example, 0.5-100 μg/ml, 1-50 μg/ml or 2-10 μg/ml.
公知のカルモジュリン阻害剤、例えば、W-7、カルミダゾリウム、ビスインドリルマレイミドI、トリフルオペラジン、ルテニウムレッド、オフィオボリンA、CaMキナーゼII(290-309)、E6ベルバミン、マストパラン、コンパウンド48/80、フェノキシベンザミン、W-7アイソマー、ポリステスマストパラン、A-7、フルフェナジン-N-2-クロロエタン、W-13、W-13アイソマー、CGS 9343B、W-5アイソマー、W-12、N-(5-アミノペンチル)-5-クロロ-2-ナフタレンスルホンアミド、W-5を、当分野で知られている方法、例えば製造業者の推奨する方法により、適宜使用してもよい。2種以上のカルモジュリン阻害剤を併用してもよい。
Known calmodulin inhibitors such as W-7, calmidazolium, bisindolylmaleimide I, trifluoperazine, ruthenium red, ophiovorin A, CaM kinase II (290-309), E6 berbamine, mastoparan, compound 48/80, phenoxy Benzamine, W-7 isomer, Polystesmastoparan, A-7, Fluphenazine-N-2-chloroethane, W-13, W-13 isomer, CGS 9343B, W-5 isomer, W-12, N-(5 -aminopentyl)-5-chloro-2-naphthalenesulfonamide, W-5, may be used as appropriate by methods known in the art, such as manufacturer's recommendations. Two or more calmodulin inhibitors may be used in combination.
ある実施態様では、PKCを活性化する条件は、(2)活性化型PKCを前記細胞に発現させ、カルモジュリン阻害剤の存在下で培養することである。
In one embodiment, the condition for activating PKC is (2) allowing the cell to express activated PKC and culturing in the presence of a calmodulin inhibitor.
「活性化型PKC」は、恒常的にキナーゼ活性を示すPKC変異体を意味する。活性化型PKCは、N末端の調節領域を欠くPKCであり得る(Molecular and Cellular Biology 19(2):1313-24, 1999)。活性化型PKCは、さらに、キナーゼ活性を増強する変異を有してもよい(PNAS 115(24):E5497-E5505. 2018)。活性化型PKCの例として、PKCδ-CA(配列番号1)およびPKCαCA-M489V(配列番号2)が挙げられる。
"Activated PKC" means a PKC mutant that constitutively exhibits kinase activity. Activated PKC can be PKC lacking the N-terminal regulatory region (Molecular and Cellular Biology 19(2):1313-24, 1999). Activated PKC may further have mutations that enhance kinase activity (PNAS 115(24):E5497-E5505. 2018). Examples of activated PKC include PKCδ-CA (SEQ ID NO: 1) and PKCαCA-M489V (SEQ ID NO: 2).
PKC変異体のキナーゼ活性は、当分野で知られている各種の方法により測定し得る。例えば、培養細胞にPKC変異体を過剰発現させ、リン酸化型基質特異的抗体を用いて基質のリン酸化レベルをウェスタンブロットで検出する方法(例えば、THE JOURNAL OF BIOLOGICAL CHEMISTRY, Vol. 279, No. 27, pp. 27986-27993, 2004)、ELISA法(例えば、Cell Death and Differentiation (2015) 22, 2078-2086)、インビトロで32P-ガンマ-ATPを用いて基質へのリン酸基の取り込みを評価する方法(例えば、THE JOURNAL OF BIOLOGICAL CHEMISTRY, Vol. 262, No. 20, pp. 9569-9573, 1987)などを使用できる。PKC kinase activity kit(Enzo Life Science, #ADI-EKS-420A)などの、キナーゼ活性を測定する各種キットを使用してもよい。
Kinase activity of a PKC mutant can be measured by various methods known in the art. For example, a method of overexpressing a PKC mutant in cultured cells and detecting the phosphorylation level of the substrate by Western blotting using a phosphorylated substrate-specific antibody (for example, THE JOURNAL OF BIOLOGICAL CHEMISTRY, Vol. 279, No. 27, pp. 27986-27993, 2004), ELISA (for example, Cell Death and Differentiation (2015) 22, 2078-2086), and in vitro incorporation of phosphate groups into substrates using 32 P-gamma-ATP. Evaluation methods (eg, THE JOURNAL OF BIOLOGICAL CHEMISTRY, Vol. 262, No. 20, pp. 9569-9573, 1987) and the like can be used. Various kits that measure kinase activity may be used, such as the PKC kinase activity kit (Enzo Life Science, #ADI-EKS-420A).
ある実施態様では、PKC変異体を発現する細胞をPKC阻害剤の存在下および非存在下で培養し、リン酸化タンパク質を特異的に認識する抗体を用いる免疫学的手法によりリン酸化タンパク質の量を測定し、PKC阻害剤によりリン酸化タンパク質の量が減少する場合に、PKC変異体が活性化型PKCであると決定し得る。免疫学的手法としては、フローサイトメトリー解析、放射性同位元素免疫測定法(RIA法)、酵素免疫固相法(ELISA法)、ウェスタンブロッティング、免疫組織染色などを例示できる。
In one embodiment, cells expressing a PKC mutant are cultured in the presence and absence of a PKC inhibitor, and the amount of phosphorylated protein is determined by an immunological method using an antibody that specifically recognizes the phosphorylated protein. A PKC mutant can be determined to be an activated form of PKC if it is measured and the amount of phosphorylated protein is reduced by a PKC inhibitor. Examples of immunological techniques include flow cytometry analysis, radioisotope immunoassay (RIA), enzyme immunoassay (ELISA), western blotting, and immunohistochemical staining.
例えば、作動可能に連結されている、プロモーター、目的のタンパク質をコードする核酸、内部リボソーム進入部位(IRES)または2A自己切断ペプチド(2Aペプチド)配列、および活性化型PKCをコードする核酸を含む発現コンストラクトを使用して、目的のタンパク質と活性化型PKCを細胞に発現させることができる。ある実施態様では、プロモーター、目的のタンパク質をコードする核酸、IRESまたは2Aペプチド配列、および活性化型PKCをコードする核酸は、この順序で作動可能に連結されている。
For example, expression comprising, operably linked, a promoter, a nucleic acid encoding a protein of interest, an internal ribosome entry site (IRES) or 2A self-cleaving peptide (2A peptide) sequence, and a nucleic acid encoding an activated PKC. Constructs can be used to express proteins of interest and activated PKC in cells. In some embodiments, a promoter, a nucleic acid encoding a protein of interest, an IRES or 2A peptide sequence, and a nucleic acid encoding activated PKC are operably linked in this order.
IRESを利用して1つの発現コンストラクトから目的のタンパク質と活性化型PKCを発現させることにより、目的のタンパク質を効率よく製造することができる。IRESは、真核生物のリボソームをmRNAへリクルートすることができるRNA領域であり、タンパク質合成のプロセスの一部として、キャップ非依存的な翻訳の開始を可能にする。多くのIRESが、ウイルスゲノムおよび真核ゲノムで同定されており、合成IRESも開発されている。
By expressing the target protein and activated PKC from a single expression construct using IRES, the target protein can be produced efficiently. An IRES is an RNA region that can recruit eukaryotic ribosomes to mRNA, allowing cap-independent initiation of translation as part of the process of protein synthesis. Many IRESs have been identified in viral and eukaryotic genomes, and synthetic IRESs have also been developed.
例えば、IRESは、エンテロウイルス(例えばヒトパピローマウイルス1、ヒトコクサッキーウイルスB);ライノウイルス(例えばヒトライノウイルス);へパトウイルス(A型肝炎ウイルス);カーディオウイルス(脳心筋炎ウイルスECMVおよびタイラー脳脊髄炎ウイルス);アフトウイルス(口蹄疫ウイルス、ウマ鼻炎Aウイルス、ウマ鼻炎Bウイルス);ぺスティウイルス(例えばウシウイルス性下痢ウイルスおよびブタコレラウイルス;ヘパシウイルス(例えばC型肝炎ウイルス)およびGBウイルスB)を含む種々のウイルスに由来するものであり得る。あるいは、IRESは、レトロウイルス科のウイルス、例えば、レンチウイルスファミリーのメンバー(例えば、シミアン免疫不全ウイルスおよびヒト免疫不全ウイルス1);BLV-HTLVレトロウイルス(例えば、ヒトT-リンホトロピックウイルス1型);および哺乳動物C型レトロウイルスファミリー(例えば、モロニーマウス白血病ウイルス、フレンドマウス白血病ウイルス、ハーベイマウス肉腫ウイルス、トリ細網内皮症ウイルス、マウス白血病ウイルス(envRNA)、ラウス肉腫ウイルス)に由来するものであり得る。真核生物のmRNAに由来するIRESとしては、例えば、BiP、ショウジョウバエのアンテナペディア(エクソンdおよびe)、c-myc、およびアポトーシスのX連結阻害剤(XIAP)遺伝子のIRESが挙げられる。また、様々な合成IRESが開発されており、例えば、De Gregorio et al. (1999) EMBO J. 75:4865-74; Owens et al. (2001) PNAS 4:1471-6;およびVenkatesan et al. (2001) Molecular and Cellular Biology 21:2826-37を参照し得る。当分野で知られているさらなるIRESについては、例えば rangueil.inserm.fr/IRESdatabase を参照し得る。ある実施態様では、脳心筋炎ウイルスECMV由来のIRESを用いる。
For example, IRES can be identified by enteroviruses (e.g. human papillomavirus 1, human coxsackievirus B); rhinoviruses (e.g. human rhinovirus); hepatoviruses (hepatitis A virus); cardioviruses (encephalomyocarditis virus ECMV and Theiler's encephalomyelitis virus) ); aphthoviruses (foot-and-mouth disease virus, equine rhinitis A virus, equine rhinitis B virus); pestiviruses (e.g. bovine viral diarrhea virus and swine fever virus; hepaciviruses (e.g. hepatitis C virus) and GB virus B); can be derived from viruses of Alternatively, the IRES is a virus of the retroviridae family, such as members of the lentivirus family (e.g., simian immunodeficiency virus and human immunodeficiency virus 1); BLV-HTLV retroviruses (e.g., human T-lymphotropic virus type 1); ); and mammalian C-type retrovirus families (e.g., Moloney murine leukemia virus, Friend murine leukemia virus, Harvey murine sarcoma virus, avian reticuloendotheliosis virus, murine leukemia virus (envRNA), Rous sarcoma virus). can be IRESs derived from eukaryotic mRNAs include, for example, the IRESs of BiP, Drosophila Antennapedia (exons d and e), c-myc, and the X-linked inhibitor of apoptosis (XIAP) gene. Various synthetic IRESs have also been developed, for example De Gregorio et al. (1999) EMBO J. 75:4865-74; Owens et al. (2001) PNAS 4:1471-6; and Venkatesan et al. (2001) Molecular and Cellular Biology 21:2826-37. For additional IRESs known in the art, see, for example, rangueil.inserm.fr/IRESdatabase. In one embodiment, an IRES from encephalomyocarditis virus ECMV is used.
2Aペプチド配列は、タンパク質の翻訳中にリボソームスキッピングを誘発する。タンパク質のアミノ酸配列中に2Aペプチド配列が存在すると、当該タンパク質は2Aペプチド配列のC末端で切断された2つのポリペプチドとして翻訳される。2Aペプチドとしては、例えば、Kim, J. H., et al., PLoS One. 6(4), e18556 (2011) に記載されるペプチドが挙げられ、例えば、P2Aペプチド(配列番号3:(GSG)ATNFSLLKQAGDVEENPGP)、T2Aペプチド(配列番号4:(GSG)EGRGSLLTCGDVEENPGP)、E2Aペプチド(配列番号5:(GSG)QCTNYALLKLAGDVESNPGP)およびF2Aぺプチド(配列番号6:(GSG)VKQTLNFDLLKLAGDVESNPGP)などが知られている(各配列中、N末端のGSGは存在してもしなくてもよい)。
The 2A peptide sequence induces ribosome skipping during protein translation. When a 2A peptide sequence is present in the amino acid sequence of a protein, the protein is translated as two polypeptides truncated at the C-terminus of the 2A peptide sequence. 2A peptides include, for example, peptides described in Kim, J. H., et al., PLoS One. 6(4), e18556 (2011); for example, P2A peptide (SEQ ID NO: 3: (GSG )ATNFSLLKQAGDVEENPGP), T2A peptide (SEQ ID NO: 4: (GSG)EGRGSLLTCGDVEENPGP), E2A peptide (SEQ ID NO: 5: (GSG)QCTNYALLKLAGDVESNPGP) and F2A peptide (SEQ ID NO: 6: (GSG)VKQTLNFDLLKLAGDVESNPGP) are known ( The N-terminal GSG may or may not be present in each sequence).
(2)の条件において、カルモジュリン阻害剤は、上記(1)と同様に使用し得る。
Under the conditions of (2), calmodulin inhibitors can be used in the same manner as in (1) above.
ある実施態様では、PKCを活性化する条件は、(3)Pro領域またはRING領域を含むペプチドをコードする核酸を前記細胞に発現させ、PKC活性化剤の存在下で培養することである。
In one embodiment, the condition for activating PKC is (3) allowing the cell to express a nucleic acid encoding a peptide containing a Pro region or a RING region and culturing in the presence of a PKC activator.
目的のタンパク質をコードする核酸およびPro領域またはRING領域を含むペプチドをコードする核酸を含む発現コンストラクトを用いることにより、目的のタンパク質を効率よく製造することができる。例えば、作動可能に連結されている、プロモーター、タンパク質をコードする核酸、および、Pro領域またはRING領域を含むペプチドをコードする核酸を含む発現コンストラクトを使用し得る。ある実施態様では、プロモーター、タンパク質をコードする核酸、および、Pro領域またはRING領域を含むペプチドをコードする核酸は、この順序で作動可能に連結されている。
The target protein can be efficiently produced by using an expression construct containing a nucleic acid encoding the target protein and a nucleic acid encoding a peptide containing the Pro region or the RING region. For example, an expression construct comprising, operably linked, a promoter, nucleic acid encoding a protein, and nucleic acid encoding a peptide comprising a Pro region or a RING region may be used. In some embodiments, a promoter, a protein-encoding nucleic acid, and a peptide-encoding nucleic acid comprising a Pro region or a RING region are operably linked in this order.
Pro領域またはRING領域を含むペプチドをコードする核酸は、ペプチドに翻訳されてもされなくてもよい。翻訳されるようにする場合、目的のタンパク質をコードする核酸とPro領域またはRING領域を含むペプチドをコードする核酸の間に、IRESまたは2Aペプチド配列を挿入し得る。IRESおよび2Aペプチド配列は、上記(2)と同様に使用し得る。例えば、作動可能に連結されている、プロモーター、タンパク質をコードする核酸、IRESまたは2Aペプチド配列、および、Pro領域またはRING領域を含むペプチドをコードする核酸を含む発現コンストラクトを使用し得る。ある実施態様では、プロモーター、タンパク質をコードする核酸、IRESまたは2Aペプチド配列、および、Pro領域またはRING領域を含むペプチドをコードする核酸は、この順序で作動可能に連結されている。
A nucleic acid encoding a peptide containing a Pro region or a RING region may or may not be translated into a peptide. When made to be translated, an IRES or 2A peptide sequence may be inserted between the nucleic acid encoding the protein of interest and the nucleic acid encoding the peptide containing the Pro region or RING region. IRES and 2A peptide sequences can be used as in (2) above. For example, an expression construct comprising, operably linked, a promoter, a nucleic acid encoding a protein, an IRES or 2A peptide sequence, and a nucleic acid encoding a peptide comprising a Pro region or a RING region may be used. In some embodiments, a promoter, a nucleic acid encoding a protein, an IRES or 2A peptide sequence, and a nucleic acid encoding a peptide comprising a Pro or RING region are operably linked in that order.
本開示において、Pro領域またはRING領域はいかなる種のものであってもよく、例えば、マウス、ラット、ハムスター、ウサギ、ネコ、イヌ、ウシ、ブタ、ヒツジ、サル、ヒト等、特にヒトに由来するものである。好ましくは、Pro領域またはRING領域は、使用する細胞と同じ種に由来するものである。
In the present disclosure, the Pro region or RING region may be of any species, e.g., mouse, rat, hamster, rabbit, cat, dog, bovine, porcine, ovine, monkey, human, etc., particularly human. It is. Preferably, the Pro region or RING region is from the same species as the cells used.
Pro領域はプロリン残基に富んだアミノ酸配列を有する領域を指し、WWドメインやSH3ドメインなどの機能性ドメインとの特異的相互作用を媒介する。Pro領域としては、反復性の短いプロリン残基に富んだアミノ酸配列、タンデムに繰り返されたプロリン残基に富んだアミノ酸配列、非反復性のプロリン残基に富んだアミノ酸配列、およびヒドロキシプロリン残基に富んだアミノ酸配列を有する領域が挙げられる。Pro領域は、これだけに限定されないが、核内タンパク質、転写因子、輸送体、チャネル、および受容体などの内在性膜タンパク質、球状タンパク質、ホルモン、神経ペプチド、ムチン、免疫グロブリン、ならびに細胞外マトリックスタンパク質を含めた種々のタンパク質に見いだすことができる。
A Pro region refers to a region having an amino acid sequence rich in proline residues and mediates specific interactions with functional domains such as WW domains and SH3 domains. Pro regions include repetitive short proline residue-rich amino acid sequences, tandemly repeated proline residue-rich amino acid sequences, non-repetitive proline residue-rich amino acid sequences, and hydroxyproline residues. regions with amino acid sequences rich in Pro regions include, but are not limited to, integral membrane proteins such as nuclear proteins, transcription factors, transporters, channels and receptors, globular proteins, hormones, neuropeptides, mucins, immunoglobulins, and extracellular matrix proteins. can be found in a variety of proteins, including
Pro領域は、いかなるタンパク質に由来してもよい。Pro領域を含むタンパク質として、例えば、PML、ARHGEF1、アグリカン-1、RALGDS、DGKK、SPATA21、ラブフィリン-3A、TEAD3、SPPL2BおよびFLJ43093が挙げられる。
The Pro region may be derived from any protein. Proteins containing the Pro region include, for example, PML, ARHGEF1, aggrecan-1, RALGDS, DGKK, SPATA21, Rabufilin-3A, TEAD3, SPPL2B and FLJ43093.
RING領域は、RINGフィンガー領域とも呼ばれ、一対の亜鉛原子に結合し、タンパク質間相互作用を媒介する。RING領域は、一般的に下記の共通配列を有する。
C-X2-C-X9-39-C-X1-3-H-X2-3-C-X2-C-X4-48-C-X2-C
{式中、Cはシステイン残基であり、Hはヒスチジン残基であり、Xは任意のアミノ酸残基である。}
これらのシステイン残基およびヒスチジン残基は、亜鉛原子との結合を介した構造を形成するために必要であり、高度に保存されている。 The RING region, also called the RING finger region, binds to a pair of zinc atoms and mediates protein-protein interactions. RING regions generally have the following consensus sequences.
CX 2 -CX 9-39 -CX 1-3 -HX 2-3 -CX 2 -CX 4-48- CX 2 -C
{Wherein, C is a cysteine residue, H is a histidine residue, and X is any amino acid residue. }
These cysteine and histidine residues are required for forming the structure via binding to the zinc atom and are highly conserved.
C-X2-C-X9-39-C-X1-3-H-X2-3-C-X2-C-X4-48-C-X2-C
{式中、Cはシステイン残基であり、Hはヒスチジン残基であり、Xは任意のアミノ酸残基である。}
これらのシステイン残基およびヒスチジン残基は、亜鉛原子との結合を介した構造を形成するために必要であり、高度に保存されている。 The RING region, also called the RING finger region, binds to a pair of zinc atoms and mediates protein-protein interactions. RING regions generally have the following consensus sequences.
CX 2 -CX 9-39 -CX 1-3 -HX 2-3 -CX 2 -CX 4-48- CX 2 -C
{Wherein, C is a cysteine residue, H is a histidine residue, and X is any amino acid residue. }
These cysteine and histidine residues are required for forming the structure via binding to the zinc atom and are highly conserved.
RING領域は、いかなるタンパク質に由来してもよい。RING領域を含むタンパク質として、例えば、TRIM13、LONRF3、TRIM47、RNF135、TRIM10、TRIM72、TRIM60、TRIM39、TRIM4、TRIM43B、TRIM43、TRIM25、TRIM26、TRIM31、HTLF、BRCA1、TRIM50、TRIM21、SSA1、TRIM5d、TRIM22、KIAA0182、TRIM65、RAG1、BFAR、Pex10、RNF8、RING2、COPI、TRIM2、TRIM3、SH3RF2、PMLおよびTRIM56が挙げられる。ある実施態様では、RING領域は、PML、TRIM3、TRIM56、COPI、Pex10、BRCA1またはHTLFのRING領域である。
The RING region may be derived from any protein. Examples of proteins containing a RING region include TRIM13, LONRF3, TRIM47, RNF135, TRIM10, TRIM72, TRIM60, TRIM39, TRIM4, TRIM43B, TRIM43, TRIM25, TRIM26, TRIM31, HTLF, BRCA1, TRIM50, TRIM21, SSA1, TRIM5d, TRIM22 , KIAA0182, TRIM65, RAG1, BFAR, Pex10, RNF8, RING2, COPI, TRIM2, TRIM3, SH3RF2, PML and TRIM56. In some embodiments, the RING region is the RING region of PML, TRIM3, TRIM56, COPI, Pex10, BRCA1 or HTLF.
ある実施態様では、Pro領域またはRING領域は、前骨髄球性白血病タンパク質(PML)に由来する。PMLは、PMLボディと呼ばれる核内構造体の組み立てに必要とされる。PMLボディは多様な機能を有し、幅広い細胞内プロセスへの関与が示唆されている。ヒトPMLには複数のアイソフォームが知られており、N末端側はすべてのアイソフォームで同一のアミノ酸配列である。
In some embodiments, the Pro region or RING region is derived from promyelocytic leukemia protein (PML). PML is required for the assembly of intranuclear structures called PML bodies. PML bodies have diverse functions and have been suggested to be involved in a wide range of intracellular processes. A plurality of isoforms are known for human PML, and the N-terminal side has the same amino acid sequence in all isoforms.
ヒト野生型(WT)PMLアイソフォーム5(Gene ID: 5371, NCBI Reference Sequence: NP_150247.2)のアミノ酸配列およびヌクレオチド配列を配列番号7および8に示す。アミノ酸配列は、560個のアミノ酸からなり、図25に示す各領域を有する。配列番号7のアミノ酸配列の第1位~第45位(配列番号9)がPro領域であり、第46位~第105位(配列番号10)がRING領域である。配列番号8のヌクレオチド配列の第1位~第135位(配列番号11)がPro領域をコードし、第136位~第315位(配列番号12)がRING領域をコードする。
The amino acid and nucleotide sequences of human wild-type (WT) PML isoform 5 (Gene ID: 5371, NCBI Reference Sequence: NP_150247.2) are shown in SEQ ID NOs: 7 and 8. The amino acid sequence consists of 560 amino acids and has each region shown in FIG. In the amino acid sequence of SEQ ID NO: 7, positions 1 to 45 (SEQ ID NO: 9) are the Pro region, and positions 46 to 105 (SEQ ID NO: 10) are the RING region. Positions 1 to 135 (SEQ ID NO: 11) of the nucleotide sequence of SEQ ID NO: 8 encode the Pro region, and positions 136 to 315 (SEQ ID NO: 12) encode the RING region.
Pro領域は、あるタンパク質のアミノ酸配列と配列番号7のアミノ酸配列を最適な状態(アミノ酸の一致が最大となる状態)にアラインメントしたときに、配列番号7の第1位~第45位の領域と一致する、当該タンパク質の領域であり得る。ある実施態様では、Pro領域は、配列番号9のアミノ酸配列と少なくとも90%以上の同一性を有するアミノ酸配列を含むか、またはそれからなる。ある実施態様では、Pro領域は、配列番号9のアミノ酸配列を含む。ある実施態様では、Pro領域は、配列番号9のアミノ酸配列からなる。ある実施態様では、Pro領域は、配列番号11のヌクレオチド配列と少なくとも90%以上の同一性を有するヌクレオチド配列により、または、それを含むヌクレオチド配列によりコードされる。ある実施態様では、Pro領域は、配列番号11のヌクレオチド配列を含むヌクレオチド配列によりコードされる。ある実施態様では、Pro領域は、配列番号11のヌクレオチド配列によりコードされる。
When the amino acid sequence of a certain protein and the amino acid sequence of SEQ ID NO: 7 are aligned in an optimal state (a state in which the amino acid match is maximized), the Pro region corresponds to the region of positions 1 to 45 of SEQ ID NO: 7. It can be the region of the protein that corresponds. In some embodiments, the Pro region comprises or consists of an amino acid sequence having at least 90% or more identity to the amino acid sequence of SEQ ID NO:9. In some embodiments, the Pro region comprises the amino acid sequence of SEQ ID NO:9. In one embodiment, the Pro region consists of the amino acid sequence of SEQ ID NO:9. In some embodiments, the Pro region is encoded by a nucleotide sequence having at least 90% or more identity to the nucleotide sequence of SEQ ID NO:11 or comprising it. In some embodiments, the Pro region is encoded by a nucleotide sequence comprising the nucleotide sequence of SEQ ID NO:11. In one embodiment, the Pro region is encoded by the nucleotide sequence of SEQ ID NO:11.
RING領域は、あるタンパク質のアミノ酸配列と配列番号7のアミノ酸配列を最適な状態にアラインメントしたときに、配列番号7の第46位~第105位の領域と一致する、当該タンパク質の領域であり得る。ある実施態様では、RING領域は、配列番号10のアミノ酸配列と少なくとも90%以上の同一性を有するアミノ酸配列を含むか、またはそれからなる。ある実施態様では、RING領域は、配列番号10のアミノ酸配列を含む。ある実施態様では、RING領域は、配列番号10のアミノ酸配列からなる。ある実施態様では、RING領域は、配列番号12のヌクレオチド配列と、少なくとも90%以上の同一性を有するヌクレオチド配列により、または、それを含むヌクレオチド配列によりコードされる。ある実施態様では、RING領域は、配列番号12のヌクレオチド配列を含むヌクレオチド配列によりコードされる。ある実施態様では、RING領域は、配列番号12のヌクレオチド配列によりコードされる。
The RING region can be a region of a protein that corresponds to the region from position 46 to position 105 of SEQ ID NO: 7 when the amino acid sequence of a protein and the amino acid sequence of SEQ ID NO: 7 are optimally aligned. . In some embodiments, the RING region comprises or consists of an amino acid sequence having at least 90% or more identity to the amino acid sequence of SEQ ID NO:10. In some embodiments, the RING region comprises the amino acid sequence of SEQ ID NO:10. In one embodiment, the RING region consists of the amino acid sequence of SEQ ID NO:10. In some embodiments, the RING region is encoded by a nucleotide sequence having at least 90% or more identity to the nucleotide sequence of SEQ ID NO:12, or by a nucleotide sequence comprising the same. In some embodiments, the RING region is encoded by a nucleotide sequence comprising the nucleotide sequence of SEQ ID NO:12. In one embodiment, the RING region is encoded by the nucleotide sequence of SEQ ID NO:12.
本開示において、ヌクレオチド配列またはアミノ酸配列の同一性とは、核酸またはタンパク質間の配列の類似の程度を意味し、比較対象の配列の領域にわたって最適な状態(ヌクレオチドまたはアミノ酸の一致が最大となる状態)にアラインメントされた2つの配列を比較することにより決定される。配列同一性の数値(%)は両方の配列に存在する同一のヌクレオチドまたはアミノ酸を決定して、適合部位の数を決定し、次いでこの適合部位の数を比較対象の配列領域内のヌクレオチドまたはアミノ酸の総数で割り、得られた数値に100をかけることにより算出される。最適なアラインメントおよび配列同一性を得るためのアルゴリズムとしては、当業者が通常利用可能な種々のアルゴリズム(例えば、BLASTアルゴリズム、FASTAアルゴリズムなど)が挙げられる。配列同一性は、例えばBLAST、FASTAなどの配列解析ソフトウェアを用いて決定され得る。
In the present disclosure, nucleotide or amino acid sequence identity refers to the degree of sequence similarity between nucleic acids or proteins, and is optimal (maximum nucleotide or amino acid identity) over the region of the sequences to be compared. ) is determined by comparing the two sequences aligned to . A numerical value (%) of sequence identity is determined by determining the number of identical nucleotides or amino acids present in both sequences to determine the number of matching sites, and then dividing this number of matching sites by nucleotides or amino acids within the sequence region being compared. It is calculated by dividing by the total number and multiplying the resulting number by 100. Algorithms for obtaining optimal alignment and sequence identity include various algorithms commonly available to those of skill in the art (eg, BLAST algorithms, FASTA algorithms, etc.). Sequence identity can be determined, for example, using sequence analysis software such as BLAST, FASTA.
(3)の条件において、PKC活性化剤は、上記(1)と同様に使用し得る。
Under the conditions of (3), the PKC activator can be used in the same manner as in (1) above.
ある実施態様では、PKCを活性化する条件は、(4)化合物Xおよび化合物#1~#7から選択される化合物またはそのエステル、塩もしくは溶媒和物の存在下で前記細胞を培養することである。これらの化合物は、上記(1)に記載したPKC活性化剤に含まれ、上記の通りに使用し得る。
In one embodiment, the condition for activating PKC is (4) culturing the cells in the presence of compound X and a compound selected from compounds # 1 to #7 or esters, salts or solvates thereof. be. These compounds are included in the PKC activators described in (1) above and can be used as described above.
化合物Xおよび化合物#1~#7は、化学合成により入手してもよく、これらを含有する植物から抽出してもよい。例えば、化合物Xは黄芫花(Lowdaphne Stringbush)から、化合物#1および#2は芫花(Lilac Daphne)から、化合物#3~#5は巴豆(Croton)から、化合物#6~#7は千金子(Caper Euphorbia)から抽出し得る。これらの化合物を含む植物加工物、例えば、植物エキスまたは抽出物を使用してもよい。
Compound X and compounds #1 to #7 may be obtained by chemical synthesis or may be extracted from plants containing them. For example, compound X is from Lowdaphne Stringbush, compounds #1 and #2 are from Lilac Daphne, compounds #3-#5 are from Croton, compounds #6-#7 are Caper Euphorbia). Plant products containing these compounds, such as plant extracts or extracts, may also be used.
ある態様では、化合物Xおよび化合物#1~#7から選択される化合物またはそのエステル、塩もしくは溶媒和物を含む、PKCを活性化するための組成物が提供される。組成物は、例えば、適当な担体、賦形剤、添加剤などを含んでもよく、他の活性成分を含んでもよい。
In one aspect, a composition is provided for activating PKC comprising a compound selected from Compound X and Compounds #1 to #7 or esters, salts or solvates thereof. The composition may contain, for example, suitable carriers, excipients, additives, etc., and may contain other active ingredients.
(1)~(4)の条件でPKCを活性化する際に、さらにヒストンデアセチラーゼ阻害剤を培地に添加してもよい。ヒストンデアセチラーゼは、クロマチン構造において主要な構成因子であるヒストンを脱アセチル化する酵素であり、遺伝子の転写制御において重要な役割を果たしている。公知のヒストンデアセチラーゼ阻害剤、例えば、トリコスタチンA、M344、酪酸塩、フェニル酪酸塩、アピシジン、バルプロ酸、BML-210、デプデシン、ロミデプシン(FK-228)、HCトキシン、オキサムフラチン、スクリプタイド、スプリトマイシン、スベロイルビス-ヒドロキサム酸、ボリノスタット、ダシノスタット(LAQ-824)、パノビノスタット(LBH-589)、ベリノスッタト(PXD-101)、酢酸フェニル、IF2357、FK-228、エンチノスタット(MS-275)、モセチノスタット(MGCD0103)またはタセジナリン(CI994)、好ましくは、酪酸ナトリウム、バルプロ酸、トリコスタチンA、ボリノスタット、アピシジン、エンチノスタットまたはタセジナリン、特に好ましくは酪酸ナトリウムを、当分野で周知の方法、例えば、製造業者の推奨する方法に従って、適宜使用し得る。例えば、酪酸ナトリウムは、0.1~10mM、0.5~5mMまたは1~3mMの濃度で使用し得る。2種以上のヒストンデアセチラーゼ阻害剤を併用してもよい。
A histone deacetylase inhibitor may be added to the medium when PKC is activated under the conditions (1) to (4). Histone deacetylase is an enzyme that deacetylates histones, which are major constituents in chromatin structure, and plays an important role in the regulation of gene transcription. known histone deacetylase inhibitors such as trichostatin A, M344, butyrate, phenylbutyrate, apicidin, valproic acid, BML-210, depudecin, romidepsin (FK-228), HC toxin, oxamflatin, scriptaid, Splitomycin, suberoyl bis-hydroxamic acid, vorinostat, dacinostat (LAQ-824), panobinostat (LBH-589), belinostat (PXD-101), phenyl acetate, IF2357, FK-228, entinostat (MS-275), mosetinostat (MGCD0103) or tacedinaline (CI994), preferably sodium butyrate, valproic acid, trichostatin A, vorinostat, apicidin, entinostat or tacedinaline, particularly preferably sodium butyrate, by methods known in the art, e.g. can be used as appropriate according to the method recommended by For example, sodium butyrate can be used at concentrations of 0.1-10 mM, 0.5-5 mM or 1-3 mM. Two or more histone deacetylase inhibitors may be used in combination.
(3)または(4)の条件でPKCを活性化する際に、さらにカルモジュリン阻害剤を培地に添加してもよい。カルモジュリン阻害剤は、上記(1)と同様に使用し得る。
A calmodulin inhibitor may be added to the medium when PKC is activated under conditions (3) or (4). A calmodulin inhibitor can be used in the same manner as in (1) above.
本開示は、また、目的の遺伝子の転写を増強する方法であって、SP1、CEBP、AP1、NF-κBおよびYY1から選択される少なくとも1つの転写因子の結合部位を含むプロモーターに作動可能に連結した当該遺伝子の核酸を含む細胞を、PKCを活性化する条件下で培養することを含む方法を提供する。PKCを活性化する条件下での培養は、タンパク質の製造方法に関して説明した(1)~(4)のいずれかである。
The disclosure also provides a method of enhancing transcription of a gene of interest, wherein the promoter is operably linked to a promoter comprising binding sites for at least one transcription factor selected from SP1, CEBP, AP1, NF-κB and YY1. A method comprising culturing a cell containing the nucleic acid of the gene of interest under conditions that activate PKC. Cultivation under conditions that activate PKC is any of (1) to (4) described with respect to the protein production method.
本開示の方法に使用し得るキットも提供される。キットに含まれる各構成要素は、各々別個に、あるいは可能であれば混合した状態で、水または適当な緩衝液中に溶解されるか、または凍結乾燥された状態で、適切な容器中に収容されて提供され得る。好適な容器には、ボトル、バイアル、試験管、チューブ、プレート等が含まれる。容器は、ガラス、プラスチック、金属などの多様な材料から形成されていてよい。容器は、ラベルを有していてもよい。キットは、さらに、使用のための説明を含む文書(例えば、書面または記憶媒体等)等の、商業的見地および使用者の見地から望ましいその他の構成要素を含んでもよい。
Also provided are kits that can be used in the methods of the present disclosure. Each component contained in the kit may be dissolved in water or a suitable buffer, or lyophilized and contained in a suitable container, either separately or, if possible, in admixture. provided. Suitable containers include bottles, vials, test tubes, tubes, plates and the like. The container may be made from a variety of materials such as glass, plastic, metal, and the like. The container may have a label. Kits may further include other components desirable from a commercial and user standpoint, such as documentation (eg, written or storage media, etc.) containing instructions for use.
さらに、本発明者らは、Pro領域またはRING領域を含むペプチドをコードする核酸を発現させることにより、CMVプロモーターの転写活性を高められることを見出した。従って、ある態様では、本願は、目的のタンパク質の製造方法であって、作動可能に連結されている、SP1、CEBP、AP1、NF-κBおよびYY1から選択される少なくとも1つの転写因子の結合部位を含むプロモーター、目的のタンパク質をコードする核酸、および、Pro領域またはRING領域を含むペプチドをコードする核酸を含む発現コンストラクトを細胞に発現させ、培養することを含む方法を提供する。この方法は、上記(3)に準じて、ただし、PKC活性化剤を使用せずに、実施できる。
Furthermore, the present inventors have found that the transcriptional activity of the CMV promoter can be enhanced by expressing a nucleic acid encoding a peptide containing a Pro region or RING region. Accordingly, in one aspect, the present application provides a method for producing a protein of interest, wherein the binding site for at least one transcription factor selected from SP1, CEBP, AP1, NF-κB and YY1 is operably linked , a nucleic acid encoding a protein of interest, and a nucleic acid encoding a peptide comprising a Pro region or a RING region. This method can be performed according to (3) above, but without using a PKC activator.
例えば、下記の実施態様が提供される。
[1]目的のタンパク質の製造方法であって、SP1、CEBP、AP1、NF-κBおよびYY1から選択される少なくとも1つの転写因子の結合部位を含むプロモーターに作動可能に連結した目的のタンパク質をコードする核酸を含む細胞を、PKCを活性化する条件下で培養することを含み、
PKCを活性化する条件下での培養が、以下のいずれかである、方法:
(1)PKC活性化剤およびカルモジュリン阻害剤の存在下で前記細胞を培養する、
(2)活性化型PKCを前記細胞に発現させ、カルモジュリン阻害剤の存在下で培養する、
(3)Pro領域またはRING領域を含むペプチドをコードする核酸を前記細胞に発現させ、PKC活性化剤の存在下で培養する、および、
(4)化合物Xおよび化合物#1~#7から選択される化合物またはそのエステル、塩もしくは溶媒和物の存在下で前記細胞を培養する。
[2]目的のタンパク質を回収する工程を含む、第1項に記載の方法。 For example, the following embodiments are provided.
[1] A method for producing a protein of interest, which encodes the protein of interest operably linked to a promoter containing a binding site for at least one transcription factor selected from SP1, CEBP, AP1, NF-κB and YY1 culturing a cell containing a nucleic acid that activates PKC,
A method, wherein culturing under conditions that activate PKC is any of the following:
(1) culturing said cells in the presence of a PKC activator and a calmodulin inhibitor;
(2) expressing activated PKC in the cells and culturing in the presence of a calmodulin inhibitor;
(3) expressing a nucleic acid encoding a peptide containing a Pro region or a RING region in said cells and culturing in the presence of a PKC activator;
(4) culturing the cells in the presence of compound X and a compound selected fromcompounds # 1 to #7 or an ester, salt or solvate thereof;
[2] The method according toitem 1, comprising a step of recovering the target protein.
[1]目的のタンパク質の製造方法であって、SP1、CEBP、AP1、NF-κBおよびYY1から選択される少なくとも1つの転写因子の結合部位を含むプロモーターに作動可能に連結した目的のタンパク質をコードする核酸を含む細胞を、PKCを活性化する条件下で培養することを含み、
PKCを活性化する条件下での培養が、以下のいずれかである、方法:
(1)PKC活性化剤およびカルモジュリン阻害剤の存在下で前記細胞を培養する、
(2)活性化型PKCを前記細胞に発現させ、カルモジュリン阻害剤の存在下で培養する、
(3)Pro領域またはRING領域を含むペプチドをコードする核酸を前記細胞に発現させ、PKC活性化剤の存在下で培養する、および、
(4)化合物Xおよび化合物#1~#7から選択される化合物またはそのエステル、塩もしくは溶媒和物の存在下で前記細胞を培養する。
[2]目的のタンパク質を回収する工程を含む、第1項に記載の方法。 For example, the following embodiments are provided.
[1] A method for producing a protein of interest, which encodes the protein of interest operably linked to a promoter containing a binding site for at least one transcription factor selected from SP1, CEBP, AP1, NF-κB and YY1 culturing a cell containing a nucleic acid that activates PKC,
A method, wherein culturing under conditions that activate PKC is any of the following:
(1) culturing said cells in the presence of a PKC activator and a calmodulin inhibitor;
(2) expressing activated PKC in the cells and culturing in the presence of a calmodulin inhibitor;
(3) expressing a nucleic acid encoding a peptide containing a Pro region or a RING region in said cells and culturing in the presence of a PKC activator;
(4) culturing the cells in the presence of compound X and a compound selected from
[2] The method according to
[3]目的の遺伝子の転写を増強する方法であって、SP1、CEBP、AP1、NF-κBおよびYY1から選択される少なくとも1つの転写因子の結合部位を含むプロモーターに作動可能に連結した当該遺伝子の核酸を含む細胞を、PKCを活性化する条件下で培養することを含み、
PKCを活性化する条件下での培養が、以下のいずれかである、方法:
(1)PKC活性化剤およびカルモジュリン阻害剤の存在下で前記細胞を培養する、
(2)活性化型PKCを前記細胞に発現させ、カルモジュリン阻害剤の存在下で培養する、
(3)Pro領域またはRING領域を含むペプチドをコードする核酸を前記細胞に発現させ、PKC活性化剤の存在下で培養する、および、
(4)化合物Xおよび化合物#1~#7から選択される化合物またはそのエステル、塩もしくは溶媒和物の存在下で前記細胞を培養する。 [3] A method for enhancing transcription of a gene of interest, wherein the gene is operably linked to a promoter comprising a binding site for at least one transcription factor selected from SP1, CEBP, AP1, NF-κB and YY1 culturing a cell containing the nucleic acid of
A method, wherein culturing under conditions that activate PKC is any of the following:
(1) culturing said cells in the presence of a PKC activator and a calmodulin inhibitor;
(2) expressing activated PKC in the cells and culturing in the presence of a calmodulin inhibitor;
(3) expressing a nucleic acid encoding a peptide containing a Pro region or a RING region in said cells and culturing in the presence of a PKC activator;
(4) culturing the cells in the presence of compound X and a compound selected fromcompounds # 1 to #7 or an ester, salt or solvate thereof;
PKCを活性化する条件下での培養が、以下のいずれかである、方法:
(1)PKC活性化剤およびカルモジュリン阻害剤の存在下で前記細胞を培養する、
(2)活性化型PKCを前記細胞に発現させ、カルモジュリン阻害剤の存在下で培養する、
(3)Pro領域またはRING領域を含むペプチドをコードする核酸を前記細胞に発現させ、PKC活性化剤の存在下で培養する、および、
(4)化合物Xおよび化合物#1~#7から選択される化合物またはそのエステル、塩もしくは溶媒和物の存在下で前記細胞を培養する。 [3] A method for enhancing transcription of a gene of interest, wherein the gene is operably linked to a promoter comprising a binding site for at least one transcription factor selected from SP1, CEBP, AP1, NF-κB and YY1 culturing a cell containing the nucleic acid of
A method, wherein culturing under conditions that activate PKC is any of the following:
(1) culturing said cells in the presence of a PKC activator and a calmodulin inhibitor;
(2) expressing activated PKC in the cells and culturing in the presence of a calmodulin inhibitor;
(3) expressing a nucleic acid encoding a peptide containing a Pro region or a RING region in said cells and culturing in the presence of a PKC activator;
(4) culturing the cells in the presence of compound X and a compound selected from
[4]プロモーターが、SP1、CEBP、AP1、NF-κBおよびYY1の結合部位を含む、第1項~第3項のいずれかに記載の方法。
[5]プロモーターが、さらにCREBの結合部位を含む、第1項~第4項のいずれかに記載の方法。
[6]プロモーターが、CMVプロモーター、CAGプロモーターまたはEF1プロモーターである、第1項~第5項のいずれかに記載の方法。
[7]プロモーターがCMVプロモーターである、第1項~第6項のいずれかに記載の方法。 [4] The method according to any one ofitems 1 to 3, wherein the promoter contains binding sites for SP1, CEBP, AP1, NF-κB and YY1.
[5] The method according to any one ofitems 1 to 4, wherein the promoter further comprises a CREB binding site.
[6] The method according to any one ofitems 1 to 5, wherein the promoter is CMV promoter, CAG promoter or EF1 promoter.
[7] The method according to any one ofitems 1 to 6, wherein the promoter is a CMV promoter.
[5]プロモーターが、さらにCREBの結合部位を含む、第1項~第4項のいずれかに記載の方法。
[6]プロモーターが、CMVプロモーター、CAGプロモーターまたはEF1プロモーターである、第1項~第5項のいずれかに記載の方法。
[7]プロモーターがCMVプロモーターである、第1項~第6項のいずれかに記載の方法。 [4] The method according to any one of
[5] The method according to any one of
[6] The method according to any one of
[7] The method according to any one of
[8]PKCを活性化する条件下での培養が、(1)PKC活性化剤およびカルモジュリン阻害剤の存在下で前記細胞を培養することである、第1項~第7項のいずれかに記載の方法。
[9]PKCを活性化する条件下での培養が、(2)活性化型PKCを前記細胞に発現させ、カルモジュリン阻害剤の存在下で培養することである、第1項~第7項のいずれかに記載の方法。
[10]前記細胞が、作動可能に連結されている、プロモーター、目的のタンパク質をコードする核酸、内部リボソーム進入部位または2Aペプチド配列、および、活性化型PKCをコードする核酸を含む発現コンストラクトを含む、第9項に記載の方法。
[11]プロモーター、目的のタンパク質をコードする核酸、内部リボソーム進入部位または2Aペプチド配列、および、活性化型PKCをコードする核酸が、この順序で作動可能に連結されている、第10項に記載の方法。
[12]活性化型PKCが、PKCδ-CAまたはPKCαCA-M489Vである、第9項~第11項のいずれかに記載の方法。 [8] Any one ofitems 1 to 7, wherein the culturing under conditions that activate PKC is (1) culturing the cells in the presence of a PKC activator and a calmodulin inhibitor. described method.
[9] The culture under conditions that activate PKC is (2) allowing the cells to express activated PKC and culturing in the presence of a calmodulin inhibitor. Any method described.
[10] The cell comprises an expression construct comprising, operably linked, a promoter, a nucleic acid encoding a protein of interest, an internal ribosome entry site or 2A peptide sequence, and a nucleic acid encoding an activated PKC. ,paragraph 9.
[11]Item 10, wherein a promoter, a nucleic acid encoding a protein of interest, an internal ribosome entry site or 2A peptide sequence, and a nucleic acid encoding activated PKC are operably linked in this order. the method of.
[12] The method according to any one ofitems 9 to 11, wherein the activated PKC is PKCδ-CA or PKCαCA-M489V.
[9]PKCを活性化する条件下での培養が、(2)活性化型PKCを前記細胞に発現させ、カルモジュリン阻害剤の存在下で培養することである、第1項~第7項のいずれかに記載の方法。
[10]前記細胞が、作動可能に連結されている、プロモーター、目的のタンパク質をコードする核酸、内部リボソーム進入部位または2Aペプチド配列、および、活性化型PKCをコードする核酸を含む発現コンストラクトを含む、第9項に記載の方法。
[11]プロモーター、目的のタンパク質をコードする核酸、内部リボソーム進入部位または2Aペプチド配列、および、活性化型PKCをコードする核酸が、この順序で作動可能に連結されている、第10項に記載の方法。
[12]活性化型PKCが、PKCδ-CAまたはPKCαCA-M489Vである、第9項~第11項のいずれかに記載の方法。 [8] Any one of
[9] The culture under conditions that activate PKC is (2) allowing the cells to express activated PKC and culturing in the presence of a calmodulin inhibitor. Any method described.
[10] The cell comprises an expression construct comprising, operably linked, a promoter, a nucleic acid encoding a protein of interest, an internal ribosome entry site or 2A peptide sequence, and a nucleic acid encoding an activated PKC. ,
[11]
[12] The method according to any one of
[13]PKCを活性化する条件下での培養が、(3)Pro領域またはRING領域を含むペプチドをコードする核酸を細胞に発現させ、PKC活性化剤の存在下で培養することである、第1項~第7項のいずれかに記載の方法。
[14]RING領域を含むペプチドをコードする核酸を細胞に発現させる、第13項に記載の方法。
[15]RING領域が、
C-X2-C-X9-39-C-X1-3-H-X2-3-C-X2-C-X4-48-C-X2-C
{式中、Cはシステイン残基であり、Hはヒスチジン残基であり、Xは任意のアミノ酸残基である。}
のアミノ酸配列を含む、第14項に記載の方法。
[16]RING領域が、TRIM13、LONRF3、TRIM47、RNF135、TRIM10、TRIM72、TRIM60、TRIM39、TRIM4、TRIM43B、TRIM43、TRIM25、TRIM26、TRIM31、HTLF、BRCA1、TRIM50、TRIM21、SSA1、TRIM5d、TRIM22、KIAA0182、TRIM65、RAG1、BFAR、Pex10、RNF8、RING2、COPI、TRIM2、TRIM3、SH3RF2、PMLまたはTRIM56のRING領域である、第14項に記載の方法。
[17]RING領域が、PML、TRIM3、TRIM56、COPI、Pex10、BRCA1またはHTLFのRING領域である、第14項~第16項のいずれかに記載の方法。
[18]RING領域がPMLのRING領域である、第14項~第17項のいずれかに記載の方法。
[19]RING領域が、配列番号10のアミノ酸配列と少なくとも90%以上の同一性を有するアミノ酸配列からなる、第14項~第18項のいずれかに記載の方法。
[20]RING領域が、配列番号12のヌクレオチド配列と、少なくとも90%以上の同一性を有するヌクレオチド配列によりコードされる、第14項~第19項のいずれかに記載の方法。 [13] Culturing under conditions that activate PKC is (3) allowing cells to express a nucleic acid encoding a peptide containing a Pro region or a RING region, and culturing in the presence of a PKC activator. 8. The method according to any one ofitems 1 to 7.
[14] The method according toitem 13, wherein a nucleic acid encoding a peptide containing a RING region is expressed in a cell.
[15] The RING area is
CX 2 -CX 9-39 -CX 1-3 -HX 2-3 -CX 2 -CX 4-48- CX 2 -C
{Wherein, C is a cysteine residue, H is a histidine residue, and X is any amino acid residue. }
15. The method ofparagraph 14, comprising the amino acid sequence of
[16] The RING region is TRIM13, LONRF3, TRIM47, RNF135, TRIM10, TRIM72, TRIM60, TRIM39, TRIM4, TRIM43B, TRIM43, TRIM25, TRIM26, TRIM31, HTLF, BRCA1, TRIM50, TRIM21, SSA1, TRIM5d, TRIM22, KIAA0182 , TRIM65, RAG1, BFAR, Pex10, RNF8, RING2, COPI, TRIM2, TRIM3, SH3RF2, PML or TRIM56.
[17] The method according to any one ofitems 14 to 16, wherein the RING region is that of PML, TRIM3, TRIM56, COPI, Pex10, BRCA1 or HTLF.
[18] The method according to any one ofitems 14 to 17, wherein the RING region is a PML RING region.
[19] The method according to any one ofitems 14 to 18, wherein the RING region consists of an amino acid sequence having at least 90% or more identity with the amino acid sequence of SEQ ID NO:10.
[20] The method according to any one ofitems 14 to 19, wherein the RING region is encoded by a nucleotide sequence having at least 90% or more identity with the nucleotide sequence of SEQ ID NO:12.
[14]RING領域を含むペプチドをコードする核酸を細胞に発現させる、第13項に記載の方法。
[15]RING領域が、
C-X2-C-X9-39-C-X1-3-H-X2-3-C-X2-C-X4-48-C-X2-C
{式中、Cはシステイン残基であり、Hはヒスチジン残基であり、Xは任意のアミノ酸残基である。}
のアミノ酸配列を含む、第14項に記載の方法。
[16]RING領域が、TRIM13、LONRF3、TRIM47、RNF135、TRIM10、TRIM72、TRIM60、TRIM39、TRIM4、TRIM43B、TRIM43、TRIM25、TRIM26、TRIM31、HTLF、BRCA1、TRIM50、TRIM21、SSA1、TRIM5d、TRIM22、KIAA0182、TRIM65、RAG1、BFAR、Pex10、RNF8、RING2、COPI、TRIM2、TRIM3、SH3RF2、PMLまたはTRIM56のRING領域である、第14項に記載の方法。
[17]RING領域が、PML、TRIM3、TRIM56、COPI、Pex10、BRCA1またはHTLFのRING領域である、第14項~第16項のいずれかに記載の方法。
[18]RING領域がPMLのRING領域である、第14項~第17項のいずれかに記載の方法。
[19]RING領域が、配列番号10のアミノ酸配列と少なくとも90%以上の同一性を有するアミノ酸配列からなる、第14項~第18項のいずれかに記載の方法。
[20]RING領域が、配列番号12のヌクレオチド配列と、少なくとも90%以上の同一性を有するヌクレオチド配列によりコードされる、第14項~第19項のいずれかに記載の方法。 [13] Culturing under conditions that activate PKC is (3) allowing cells to express a nucleic acid encoding a peptide containing a Pro region or a RING region, and culturing in the presence of a PKC activator. 8. The method according to any one of
[14] The method according to
[15] The RING area is
CX 2 -CX 9-39 -CX 1-3 -HX 2-3 -CX 2 -CX 4-48- CX 2 -C
{Wherein, C is a cysteine residue, H is a histidine residue, and X is any amino acid residue. }
15. The method of
[16] The RING region is TRIM13, LONRF3, TRIM47, RNF135, TRIM10, TRIM72, TRIM60, TRIM39, TRIM4, TRIM43B, TRIM43, TRIM25, TRIM26, TRIM31, HTLF, BRCA1, TRIM50, TRIM21, SSA1, TRIM5d, TRIM22, KIAA0182 , TRIM65, RAG1, BFAR, Pex10, RNF8, RING2, COPI, TRIM2, TRIM3, SH3RF2, PML or TRIM56.
[17] The method according to any one of
[18] The method according to any one of
[19] The method according to any one of
[20] The method according to any one of
[21]Pro領域を含むペプチドをコードする核酸を細胞に発現させる、第13項に記載の方法。
[22]Pro領域が、PML、ARHGEF1、アグリカン-1、RALGDS、DGKK、SPATA21、ラブフィリン-3A、TEAD3、SPPL2BまたはFLJ43093のPro領域である、第21項に記載の方法。
[23]Pro領域がPMLのPro領域である、第21項または第22項に記載の方法。
[24]Pro領域が、配列番号9のアミノ酸配列と少なくとも90%以上の同一性を有するアミノ酸配列からなる、第21項~第23項のいずれかに記載の方法。
[25]Pro領域を含むペプチドをコードする核酸が、配列番号11のヌクレオチド配列と、少なくとも90%以上の同一性を有するヌクレオチド配列からなる、第21項~第24項のいずれかに記載の方法。
[26]Pro領域およびRING領域を含むペプチドをコードする核酸を細胞に発現させる、第13項~第25項のいずれかに記載の方法。 [21] The method according toitem 13, wherein a nucleic acid encoding a peptide containing a Pro region is expressed in a cell.
[22] The method according to item 21, wherein the Pro region is the Pro region of PML, ARHGEF1, aggrecan-1, RALGDS, DGKK, SPATA21, Rabufilin-3A, TEAD3, SPPL2B or FLJ43093.
[23] The method according toitem 21 or 22, wherein the Pro region is a PML Pro region.
[24] The method according to any one of items 21 to 23, wherein the Pro region consists of an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO:9.
[25] The method according to any one of items 21 to 24, wherein the nucleic acid encoding the peptide containing the Pro region consists of a nucleotide sequence having at least 90% identity with the nucleotide sequence of SEQ ID NO: 11. .
[26] The method according to any one ofitems 13 to 25, wherein a nucleic acid encoding a peptide containing a Pro region and a RING region is expressed in a cell.
[22]Pro領域が、PML、ARHGEF1、アグリカン-1、RALGDS、DGKK、SPATA21、ラブフィリン-3A、TEAD3、SPPL2BまたはFLJ43093のPro領域である、第21項に記載の方法。
[23]Pro領域がPMLのPro領域である、第21項または第22項に記載の方法。
[24]Pro領域が、配列番号9のアミノ酸配列と少なくとも90%以上の同一性を有するアミノ酸配列からなる、第21項~第23項のいずれかに記載の方法。
[25]Pro領域を含むペプチドをコードする核酸が、配列番号11のヌクレオチド配列と、少なくとも90%以上の同一性を有するヌクレオチド配列からなる、第21項~第24項のいずれかに記載の方法。
[26]Pro領域およびRING領域を含むペプチドをコードする核酸を細胞に発現させる、第13項~第25項のいずれかに記載の方法。 [21] The method according to
[22] The method according to item 21, wherein the Pro region is the Pro region of PML, ARHGEF1, aggrecan-1, RALGDS, DGKK, SPATA21, Rabufilin-3A, TEAD3, SPPL2B or FLJ43093.
[23] The method according to
[24] The method according to any one of items 21 to 23, wherein the Pro region consists of an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO:9.
[25] The method according to any one of items 21 to 24, wherein the nucleic acid encoding the peptide containing the Pro region consists of a nucleotide sequence having at least 90% identity with the nucleotide sequence of SEQ ID NO: 11. .
[26] The method according to any one of
[27]前記細胞が、作動可能に連結されている、プロモーター、目的のタンパク質をコードする核酸、および、Pro領域またはRING領域を含むペプチドをコードする核酸を含む発現コンストラクトを含む、第13項~第26項のいずれかに記載の方法。
[28]プロモーター、目的のタンパク質をコードする核酸、および、Pro領域またはRING領域を含むペプチドをコードする核酸が、この順序で作動可能に連結されている、第27項に記載の方法。
[29]発現コンストラクトがさらに内部リボソーム進入部位または2Aペプチド配列をコードする核酸を含む、第27項または第28項に記載の方法。
[30]プロモーター、目的のタンパク質をコードする核酸、内部リボソーム進入部位または2Aペプチド配列、および、Pro領域またはRING領域を含むペプチドをコードする核酸が、この順序で作動可能に連結されている、第27項~第29項のいずれかに記載の方法。
[31]カルモジュリン阻害剤の存在下で細胞を培養する、第13項~第30項のいずれかに記載の方法。 [27] Items 13-, wherein the cell comprises an expression construct comprising, operably linked, a promoter, a nucleic acid encoding a protein of interest, and a nucleic acid encoding a peptide comprising a Pro region or a RING region. 27. The method of any of clauses 26.
[28] The method according to item 27, wherein a promoter, a nucleic acid encoding a protein of interest, and a nucleic acid encoding a peptide containing a Pro region or a RING region are operably linked in this order.
[29] The method of paragraphs 27 or 28, wherein the expression construct further comprises a nucleic acid encoding an internal ribosome entry site or 2A peptide sequence.
[30] A promoter, a nucleic acid encoding a protein of interest, an internal ribosome entry site or 2A peptide sequence, and a nucleic acid encoding a peptide containing a Pro region or RING region are operably linked in this order. 29. The method according to any one of paragraphs 27-29.
[31] The method according to any one ofitems 13 to 30, wherein the cell is cultured in the presence of a calmodulin inhibitor.
[28]プロモーター、目的のタンパク質をコードする核酸、および、Pro領域またはRING領域を含むペプチドをコードする核酸が、この順序で作動可能に連結されている、第27項に記載の方法。
[29]発現コンストラクトがさらに内部リボソーム進入部位または2Aペプチド配列をコードする核酸を含む、第27項または第28項に記載の方法。
[30]プロモーター、目的のタンパク質をコードする核酸、内部リボソーム進入部位または2Aペプチド配列、および、Pro領域またはRING領域を含むペプチドをコードする核酸が、この順序で作動可能に連結されている、第27項~第29項のいずれかに記載の方法。
[31]カルモジュリン阻害剤の存在下で細胞を培養する、第13項~第30項のいずれかに記載の方法。 [27] Items 13-, wherein the cell comprises an expression construct comprising, operably linked, a promoter, a nucleic acid encoding a protein of interest, and a nucleic acid encoding a peptide comprising a Pro region or a RING region. 27. The method of any of clauses 26.
[28] The method according to item 27, wherein a promoter, a nucleic acid encoding a protein of interest, and a nucleic acid encoding a peptide containing a Pro region or a RING region are operably linked in this order.
[29] The method of paragraphs 27 or 28, wherein the expression construct further comprises a nucleic acid encoding an internal ribosome entry site or 2A peptide sequence.
[30] A promoter, a nucleic acid encoding a protein of interest, an internal ribosome entry site or 2A peptide sequence, and a nucleic acid encoding a peptide containing a Pro region or RING region are operably linked in this order. 29. The method according to any one of paragraphs 27-29.
[31] The method according to any one of
[32]PKC活性化剤が、式(I)、式(II)または式(III)の化合物またはそのエステル、塩もしくは溶媒和物である、第8項および第13項~第31項のいずれかに記載の方法。
[33]PKC活性化剤が、式(I)の化合物またはそのエステル、塩もしくは溶媒和物である、第32項に記載の方法。
[34]R1が、Hまたは-OC(O)R3であり、R2が、C6-12アルキルまたはC6-12アルケニルであり、R3が、C1-6アルキルまたはC6-14アリールである、第33項に記載の方法。
[35]R1が、Hまたは-OC(O)R3であり、R2が、ノニルまたは1,3-ノナジエニルであり、R3が、メチルまたはフェニルである、第33項または第34項に記載の方法。
[36]PKC活性化剤が、式(II)の化合物またはそのエステル、塩もしくは溶媒和物である、第32項に記載の方法。
[37]R4が、Hまたは-OC(O)R6であり、R5が、C1-6アルキルまたはC2-6アルケニルであり、R6が、C1-18アルキルである、第36項に記載の方法。
[38]R4が、Hまたは-OC(O)R6であり、R5が、メチル、プロピル、sec-ブチルまたはブテニルであり、R6が、プロピル、ノニルまたはトリデシルである、第36項または第37項に記載の方法。
[39]PKC活性化剤が、式(III)の化合物またはそのエステル、塩もしくは溶媒和物である、第32項に記載の方法。
[40]R7が、Hまたは-C(O)R9であり、R8が、Hまたは-C(O)R9であり、R9が、C1-18アルキルまたはC2-18アルケニルである、第39項に記載の方法。
[41]R7が、Hまたは-C(O)R9であり、R8が、Hまたは-C(O)R9であり、R9が、ペンタデシルまたはブテニルである、第39項または第40項に記載の方法。
[42]PKC活性化剤が、化合物X、化合物#1~#7、TPA、プロストラチン、(-)-インドラクタムV、ホルボール12,13-ジブチラート、インゲノール3-アンゲラートおよび(2S,5S)-(E,E)-8-(5-(4-(トリフルオロメチル)フェニル)-2,4-ペンタジエノイルアミノ)ベンゾラクタムから選択される化合物またはそのエステル、塩もしくは溶媒和物である、第8項および第13項~第31項のいずれかに記載の方法。
[43]PKC活性化剤が、化合物Xおよび化合物#1~#7から選択される化合物またはそのエステル、塩もしくは溶媒和物である、第42項に記載の方法。
[44]PKC活性化剤が、化合物Xまたはそのエステル、塩もしくは溶媒和物である、第42項または第43項に記載の方法。 [32] Any ofparagraphs 8 and 13-31, wherein the PKC activator is a compound of formula (I), formula (II) or formula (III) or an ester, salt or solvate thereof The method described in Crab.
[33] The method of paragraph 32, wherein the PKC activator is a compound of formula (I) or an ester, salt or solvate thereof.
[34] R 1 is H or —OC(O)R 3 , R 2 is C 6-12 alkyl or C 6-12 alkenyl, R 3 is C 1-6 alkyl or C 6- 34. The method of paragraph 33, which is 14 aryl.
[35] Item 33 or 34, wherein R 1 is H or —OC(O)R 3 , R 2 is nonyl or 1,3-nonadienyl, and R 3 is methyl or phenyl The method described in .
[36] The method of paragraph 32, wherein the PKC activator is a compound of formula (II) or an ester, salt or solvate thereof.
[37] R 4 is H or —OC(O)R 6 , R 5 is C 1-6 alkyl or C 2-6 alkenyl, and R 6 is C 1-18 alkyl; 36. The method of clause 36.
[38] Paragraph 36 wherein R 4 is H or —OC(O)R 6 , R 5 is methyl, propyl, sec-butyl or butenyl and R 6 is propyl, nonyl or tridecyl Or the method ofclause 37.
[39] The method of paragraph 32, wherein the PKC activator is a compound of formula (III) or an ester, salt or solvate thereof.
[40] R 7 is H or —C(O)R 9 , R 8 is H or —C(O)R 9 and R 9 is C 1-18 alkyl or C 2-18 alkenyl 40. The method of clause 39, wherein
[41] Item 39 or wherein R 7 is H or -C(O)R 9 , R 8 is H or -C(O)R 9 and R 9 is pentadecyl orbutenyl 40. The method of paragraph 40.
[42] The PKC activator is compound X, compounds #1-#7, TPA, prostratin, (-)-indolactam V,phorbol 12,13-dibutyrate, ingenol 3-angelate and (2S,5S)- (E,E)-8-(5-(4-(trifluoromethyl)phenyl)-2,4-pentadienoylamino)benzolactam or an ester, salt or solvate thereof; 32. The method of any of paragraphs 8 and 13-31.
[43] The method of paragraph 42, wherein the PKC activator is a compound selected from Compound X and Compounds #1 to #7 or an ester, salt or solvate thereof.
[44] The method of paragraph 42 or 43, wherein the PKC activator is compound X or an ester, salt or solvate thereof.
[33]PKC活性化剤が、式(I)の化合物またはそのエステル、塩もしくは溶媒和物である、第32項に記載の方法。
[34]R1が、Hまたは-OC(O)R3であり、R2が、C6-12アルキルまたはC6-12アルケニルであり、R3が、C1-6アルキルまたはC6-14アリールである、第33項に記載の方法。
[35]R1が、Hまたは-OC(O)R3であり、R2が、ノニルまたは1,3-ノナジエニルであり、R3が、メチルまたはフェニルである、第33項または第34項に記載の方法。
[36]PKC活性化剤が、式(II)の化合物またはそのエステル、塩もしくは溶媒和物である、第32項に記載の方法。
[37]R4が、Hまたは-OC(O)R6であり、R5が、C1-6アルキルまたはC2-6アルケニルであり、R6が、C1-18アルキルである、第36項に記載の方法。
[38]R4が、Hまたは-OC(O)R6であり、R5が、メチル、プロピル、sec-ブチルまたはブテニルであり、R6が、プロピル、ノニルまたはトリデシルである、第36項または第37項に記載の方法。
[39]PKC活性化剤が、式(III)の化合物またはそのエステル、塩もしくは溶媒和物である、第32項に記載の方法。
[40]R7が、Hまたは-C(O)R9であり、R8が、Hまたは-C(O)R9であり、R9が、C1-18アルキルまたはC2-18アルケニルである、第39項に記載の方法。
[41]R7が、Hまたは-C(O)R9であり、R8が、Hまたは-C(O)R9であり、R9が、ペンタデシルまたはブテニルである、第39項または第40項に記載の方法。
[42]PKC活性化剤が、化合物X、化合物#1~#7、TPA、プロストラチン、(-)-インドラクタムV、ホルボール12,13-ジブチラート、インゲノール3-アンゲラートおよび(2S,5S)-(E,E)-8-(5-(4-(トリフルオロメチル)フェニル)-2,4-ペンタジエノイルアミノ)ベンゾラクタムから選択される化合物またはそのエステル、塩もしくは溶媒和物である、第8項および第13項~第31項のいずれかに記載の方法。
[43]PKC活性化剤が、化合物Xおよび化合物#1~#7から選択される化合物またはそのエステル、塩もしくは溶媒和物である、第42項に記載の方法。
[44]PKC活性化剤が、化合物Xまたはそのエステル、塩もしくは溶媒和物である、第42項または第43項に記載の方法。 [32] Any of
[33] The method of paragraph 32, wherein the PKC activator is a compound of formula (I) or an ester, salt or solvate thereof.
[34] R 1 is H or —OC(O)R 3 , R 2 is C 6-12 alkyl or C 6-12 alkenyl, R 3 is C 1-6 alkyl or C 6- 34. The method of paragraph 33, which is 14 aryl.
[35] Item 33 or 34, wherein R 1 is H or —OC(O)R 3 , R 2 is nonyl or 1,3-nonadienyl, and R 3 is methyl or phenyl The method described in .
[36] The method of paragraph 32, wherein the PKC activator is a compound of formula (II) or an ester, salt or solvate thereof.
[37] R 4 is H or —OC(O)R 6 , R 5 is C 1-6 alkyl or C 2-6 alkenyl, and R 6 is C 1-18 alkyl; 36. The method of clause 36.
[38] Paragraph 36 wherein R 4 is H or —OC(O)R 6 , R 5 is methyl, propyl, sec-butyl or butenyl and R 6 is propyl, nonyl or tridecyl Or the method of
[39] The method of paragraph 32, wherein the PKC activator is a compound of formula (III) or an ester, salt or solvate thereof.
[40] R 7 is H or —C(O)R 9 , R 8 is H or —C(O)R 9 and R 9 is C 1-18 alkyl or C 2-18 alkenyl 40. The method of clause 39, wherein
[41] Item 39 or wherein R 7 is H or -C(O)R 9 , R 8 is H or -C(O)R 9 and R 9 is pentadecyl or
[42] The PKC activator is compound X, compounds #1-#7, TPA, prostratin, (-)-indolactam V,
[43] The method of paragraph 42, wherein the PKC activator is a compound selected from Compound X and Compounds #1 to #7 or an ester, salt or solvate thereof.
[44] The method of paragraph 42 or 43, wherein the PKC activator is compound X or an ester, salt or solvate thereof.
[45]PKCを活性化する条件下での培養が、(4)化合物Xおよび化合物#1~#7から選択される化合物またはそのエステル、塩もしくは溶媒和物の存在下で前記細胞を培養することである、第1項~第7項のいずれかに記載の方法。
[46]化合物Xまたはそのエステル、塩もしくは溶媒和物の存在下で前記細胞を培養する、第45項に記載の方法。
[47]カルモジュリン阻害剤の存在下で細胞を培養する、第45項または第46項に記載の方法。
[48]カルモジュリン阻害剤が、式(IV)の化合物またはそのエステル、塩もしくは溶媒和物である、第8項~第12項、第31項および第47項のいずれかに記載の方法。
[49]カルモジュリン阻害剤が、SC-9、SC-10またはW-7である、第48項に記載の方法。
[50]カルモジュリン阻害剤がSC-10である、第48項または第49項に記載の方法。
[51]ヒストンデアセチラーゼ阻害剤の存在下で細胞を培養する、第1項~第50項のいずれかに記載の方法。
[52]ヒストンデアセチラーゼ阻害剤が、酪酸ナトリウム、バルプロ酸、トリコスタチンA、ボリノスタット、アピシジン、エンチノスタットまたはタセジナリンである、第51項に記載の方法。
[53]ヒストンデアセチラーゼ阻害剤が酪酸ナトリウムである、第51項または第52項に記載の方法。 [45] culturing under conditions that activate PKC comprises (4) culturing said cells in the presence of a compound selected from compound X and compounds #1 to #7 or an ester, salt orsolvate thereof 8. The method according to any one of paragraphs 1 to 7, wherein
[46] The method ofparagraph 45, wherein the cell is cultured in the presence of compound X or its ester, salt or solvate.
[47] The method of paragraph 45 or 46, wherein the cell is cultured in the presence of a calmodulin inhibitor.
[48] The method of any one of paragraphs 8-12, 31 and 47, wherein the calmodulin inhibitor is a compound of formula (IV) or an ester, salt or solvate thereof.
[49] The method of paragraph 48, wherein the calmodulin inhibitor is SC-9, SC-10 or W-7.
[50] The method of paragraph 48 orparagraph 49, wherein the calmodulin inhibitor is SC-10.
[51] The method according to any one ofitems 1 to 50, wherein the cell is cultured in the presence of a histone deacetylase inhibitor.
[52] The method of paragraph 51, wherein the histone deacetylase inhibitor is sodium butyrate, valproic acid, trichostatin A, vorinostat, apicidin, entinostat or tacedinaline.
[53] The method of paragraph 51 or 52, wherein the histone deacetylase inhibitor is sodium butyrate.
[46]化合物Xまたはそのエステル、塩もしくは溶媒和物の存在下で前記細胞を培養する、第45項に記載の方法。
[47]カルモジュリン阻害剤の存在下で細胞を培養する、第45項または第46項に記載の方法。
[48]カルモジュリン阻害剤が、式(IV)の化合物またはそのエステル、塩もしくは溶媒和物である、第8項~第12項、第31項および第47項のいずれかに記載の方法。
[49]カルモジュリン阻害剤が、SC-9、SC-10またはW-7である、第48項に記載の方法。
[50]カルモジュリン阻害剤がSC-10である、第48項または第49項に記載の方法。
[51]ヒストンデアセチラーゼ阻害剤の存在下で細胞を培養する、第1項~第50項のいずれかに記載の方法。
[52]ヒストンデアセチラーゼ阻害剤が、酪酸ナトリウム、バルプロ酸、トリコスタチンA、ボリノスタット、アピシジン、エンチノスタットまたはタセジナリンである、第51項に記載の方法。
[53]ヒストンデアセチラーゼ阻害剤が酪酸ナトリウムである、第51項または第52項に記載の方法。 [45] culturing under conditions that activate PKC comprises (4) culturing said cells in the presence of a compound selected from compound X and compounds #1 to #7 or an ester, salt or
[46] The method of
[47] The method of
[48] The method of any one of paragraphs 8-12, 31 and 47, wherein the calmodulin inhibitor is a compound of formula (IV) or an ester, salt or solvate thereof.
[49] The method of paragraph 48, wherein the calmodulin inhibitor is SC-9, SC-10 or W-7.
[50] The method of paragraph 48 or
[51] The method according to any one of
[52] The method of paragraph 51, wherein the histone deacetylase inhibitor is sodium butyrate, valproic acid, trichostatin A, vorinostat, apicidin, entinostat or tacedinaline.
[53] The method of paragraph 51 or 52, wherein the histone deacetylase inhibitor is sodium butyrate.
[54]目的のタンパク質を製造するためのキットであって、
(1)PKC活性化剤およびカルモジュリン阻害剤、
(2)活性化型PKCをコードする核酸およびカルモジュリン阻害剤、
(3)PKC活性化剤およびPro領域またはRING領域を含むペプチドをコードする核酸、または、
(4)化合物Xおよび化合物#1~#7から選択される化合物またはそのエステル、塩もしくは溶媒和物、
を含み、
目的のタンパク質の製造が、SP1、CEBP、AP1、NF-κBおよびYY1から選択される少なくとも1つの転写因子の結合部位を含むプロモーターに作動可能に連結した目的のタンパク質をコードする核酸を含む細胞を培養することを含む、キット。
[55]作動可能に連結されている、SP1、CEBP、AP1、NF-κBおよびYY1から選択される少なくとも1つの転写因子の結合部位を含むプロモーター、タンパク質をコードする核酸、内部リボソーム進入部位または2Aペプチド配列、および、活性化型PKCをコードする核酸を含む、発現コンストラクト。
[56]プロモーター、目的のタンパク質をコードする核酸、内部リボソーム進入部位または2Aペプチド配列、および、活性化型PKCをコードする核酸が、この順序で作動可能に連結されている、第55項に記載の発現コンストラクト。
[57]作動可能に連結されている、SP1、CEBP、AP1、NF-κBおよびYY1から選択される少なくとも1つの転写因子の結合部位を含むプロモーター、タンパク質をコードする核酸、および、Pro領域またはRING領域を含むペプチドをコードする核酸を含む、発現コンストラクト。
[58]プロモーター、タンパク質をコードする核酸、および、Pro領域またはRING領域を含むペプチドをコードする核酸が、この順序で作動可能に連結されている、第57項に記載の発現コンストラクト。
[59]内部リボソーム進入部位または2Aペプチド配列をコードする核酸を含む、第57項または第58項に記載の発現コンストラクト。
[60]プロモーター、目的のタンパク質をコードする核酸、内部リボソーム進入部位または2Aペプチド配列、および、Pro領域またはRING領域を含むペプチドをコードする核酸が、この順序で作動可能に連結されている、第57項~第59項のいずれかに記載の発現コンストラクト。
[61]化合物Xおよび化合物#1~#7から選択される化合物またはそのエステル、塩もしくは溶媒和物を含む、PKCを活性化するための組成物。 [54] A kit for producing a protein of interest, comprising:
(1) PKC activators and calmodulin inhibitors,
(2) a nucleic acid encoding activated PKC and a calmodulin inhibitor;
(3) a nucleic acid encoding a peptide comprising a PKC activator and a Pro region or a RING region, or
(4) a compound selected from compound X and compounds #1 to #7 or an ester, salt or solvate thereof;
including
Production of the protein of interest comprises a cell comprising a nucleic acid encoding the protein of interest operably linked to a promoter comprising a binding site for at least one transcription factor selected from SP1, CEBP, AP1, NF-κB and YY1. A kit comprising culturing.
[55] a promoter comprising a binding site for at least one transcription factor selected from SP1, CEBP, AP1, NF-κB and YY1, operably linked, a nucleic acid encoding a protein, an internal ribosome entry site or 2A An expression construct comprising a peptide sequence and a nucleic acid encoding activated PKC.
[56] Paragraph 55, wherein a promoter, a nucleic acid encoding a protein of interest, an internal ribosome entry site or 2A peptide sequence, and a nucleic acid encoding activated PKC are operably linked in this order. expression construct.
[57] a promoter comprising a binding site for at least one transcription factor selected from SP1, CEBP, AP1, NF-κB and YY1, operably linked, a nucleic acid encoding a protein, and a Pro region or RING An expression construct comprising a nucleic acid encoding a peptide containing region.
[58] The expression construct of paragraph 57, wherein a promoter, a protein-encoding nucleic acid, and a peptide-encoding nucleic acid comprising a Pro region or a RING region are operably linked in this order.
[59] The expression construct of paragraphs 57 or 58, comprising a nucleic acid encoding an internal ribosome entry site or 2A peptide sequence.
[60] A promoter, a nucleic acid encoding a protein of interest, an internal ribosome entry site or 2A peptide sequence, and a nucleic acid encoding a peptide containing a Pro region or RING region are operably linked in this order, 59. The expression construct of any one of paragraphs 57-59.
[61] A composition for activating PKC, comprising compound X and a compound selected fromcompounds # 1 to #7 or an ester, salt or solvate thereof.
(1)PKC活性化剤およびカルモジュリン阻害剤、
(2)活性化型PKCをコードする核酸およびカルモジュリン阻害剤、
(3)PKC活性化剤およびPro領域またはRING領域を含むペプチドをコードする核酸、または、
(4)化合物Xおよび化合物#1~#7から選択される化合物またはそのエステル、塩もしくは溶媒和物、
を含み、
目的のタンパク質の製造が、SP1、CEBP、AP1、NF-κBおよびYY1から選択される少なくとも1つの転写因子の結合部位を含むプロモーターに作動可能に連結した目的のタンパク質をコードする核酸を含む細胞を培養することを含む、キット。
[55]作動可能に連結されている、SP1、CEBP、AP1、NF-κBおよびYY1から選択される少なくとも1つの転写因子の結合部位を含むプロモーター、タンパク質をコードする核酸、内部リボソーム進入部位または2Aペプチド配列、および、活性化型PKCをコードする核酸を含む、発現コンストラクト。
[56]プロモーター、目的のタンパク質をコードする核酸、内部リボソーム進入部位または2Aペプチド配列、および、活性化型PKCをコードする核酸が、この順序で作動可能に連結されている、第55項に記載の発現コンストラクト。
[57]作動可能に連結されている、SP1、CEBP、AP1、NF-κBおよびYY1から選択される少なくとも1つの転写因子の結合部位を含むプロモーター、タンパク質をコードする核酸、および、Pro領域またはRING領域を含むペプチドをコードする核酸を含む、発現コンストラクト。
[58]プロモーター、タンパク質をコードする核酸、および、Pro領域またはRING領域を含むペプチドをコードする核酸が、この順序で作動可能に連結されている、第57項に記載の発現コンストラクト。
[59]内部リボソーム進入部位または2Aペプチド配列をコードする核酸を含む、第57項または第58項に記載の発現コンストラクト。
[60]プロモーター、目的のタンパク質をコードする核酸、内部リボソーム進入部位または2Aペプチド配列、および、Pro領域またはRING領域を含むペプチドをコードする核酸が、この順序で作動可能に連結されている、第57項~第59項のいずれかに記載の発現コンストラクト。
[61]化合物Xおよび化合物#1~#7から選択される化合物またはそのエステル、塩もしくは溶媒和物を含む、PKCを活性化するための組成物。 [54] A kit for producing a protein of interest, comprising:
(1) PKC activators and calmodulin inhibitors,
(2) a nucleic acid encoding activated PKC and a calmodulin inhibitor;
(3) a nucleic acid encoding a peptide comprising a PKC activator and a Pro region or a RING region, or
(4) a compound selected from compound X and compounds #1 to #7 or an ester, salt or solvate thereof;
including
Production of the protein of interest comprises a cell comprising a nucleic acid encoding the protein of interest operably linked to a promoter comprising a binding site for at least one transcription factor selected from SP1, CEBP, AP1, NF-κB and YY1. A kit comprising culturing.
[55] a promoter comprising a binding site for at least one transcription factor selected from SP1, CEBP, AP1, NF-κB and YY1, operably linked, a nucleic acid encoding a protein, an internal ribosome entry site or 2A An expression construct comprising a peptide sequence and a nucleic acid encoding activated PKC.
[56] Paragraph 55, wherein a promoter, a nucleic acid encoding a protein of interest, an internal ribosome entry site or 2A peptide sequence, and a nucleic acid encoding activated PKC are operably linked in this order. expression construct.
[57] a promoter comprising a binding site for at least one transcription factor selected from SP1, CEBP, AP1, NF-κB and YY1, operably linked, a nucleic acid encoding a protein, and a Pro region or RING An expression construct comprising a nucleic acid encoding a peptide containing region.
[58] The expression construct of paragraph 57, wherein a promoter, a protein-encoding nucleic acid, and a peptide-encoding nucleic acid comprising a Pro region or a RING region are operably linked in this order.
[59] The expression construct of paragraphs 57 or 58, comprising a nucleic acid encoding an internal ribosome entry site or 2A peptide sequence.
[60] A promoter, a nucleic acid encoding a protein of interest, an internal ribosome entry site or 2A peptide sequence, and a nucleic acid encoding a peptide containing a Pro region or RING region are operably linked in this order, 59. The expression construct of any one of paragraphs 57-59.
[61] A composition for activating PKC, comprising compound X and a compound selected from
本明細書で引用するすべての文献は、出典明示により本明細書の一部とする。
以下、実施例にて、本発明をさらに詳細に説明するが、本発明はこの実施例に限定されない。また、上記の説明は、すべて非限定的なものであり、本発明は添付の特許請求の範囲において定義され、その技術的思想を逸脱しない範囲で種々の変更が可能である。 All documents cited herein are hereby incorporated by reference.
EXAMPLES The present invention will be described in more detail below with reference to Examples, but the present invention is not limited to these Examples. Moreover, the above description is all non-limiting, and the present invention is defined in the appended claims, and various modifications are possible without departing from the technical idea thereof.
以下、実施例にて、本発明をさらに詳細に説明するが、本発明はこの実施例に限定されない。また、上記の説明は、すべて非限定的なものであり、本発明は添付の特許請求の範囲において定義され、その技術的思想を逸脱しない範囲で種々の変更が可能である。 All documents cited herein are hereby incorporated by reference.
EXAMPLES The present invention will be described in more detail below with reference to Examples, but the present invention is not limited to these Examples. Moreover, the above description is all non-limiting, and the present invention is defined in the appended claims, and various modifications are possible without departing from the technical idea thereof.
コンストラクト
CMVプロモーターによるルシフェラーゼ活性測定用には、pRL-CMV(Promega, #E2261)を使用した。CAGプロモーターはpCAGGSベクター、EF1プロモーターはpEFBOSベクター由来のプロモーターを使用し、pRL-CMVのCMV領域と載せ換えたコンストラクトを作製した。マウスIgG抗体(重鎖、軽鎖)、ヒトプロインスリンおよびヒトレプチン発現コンストラクトはpcDNAベクターのCMVプロモーターの下流にこれらのcDNAを挿入したコンストラクトを作製した。 For luciferase activity measurements with the construct CMV promoter, pRL-CMV (Promega, #E2261) was used. Using the pCAGGS vector as the CAG promoter and the pEFBOS vector as the EF1 promoter, a construct was prepared in which the CMV region of pRL-CMV was replaced. Mouse IgG antibody (heavy chain, light chain), human proinsulin and human leptin expression constructs were prepared by inserting these cDNAs downstream of the CMV promoter of a pcDNA vector.
CMVプロモーターによるルシフェラーゼ活性測定用には、pRL-CMV(Promega, #E2261)を使用した。CAGプロモーターはpCAGGSベクター、EF1プロモーターはpEFBOSベクター由来のプロモーターを使用し、pRL-CMVのCMV領域と載せ換えたコンストラクトを作製した。マウスIgG抗体(重鎖、軽鎖)、ヒトプロインスリンおよびヒトレプチン発現コンストラクトはpcDNAベクターのCMVプロモーターの下流にこれらのcDNAを挿入したコンストラクトを作製した。 For luciferase activity measurements with the construct CMV promoter, pRL-CMV (Promega, #E2261) was used. Using the pCAGGS vector as the CAG promoter and the pEFBOS vector as the EF1 promoter, a construct was prepared in which the CMV region of pRL-CMV was replaced. Mouse IgG antibody (heavy chain, light chain), human proinsulin and human leptin expression constructs were prepared by inserting these cDNAs downstream of the CMV promoter of a pcDNA vector.
細胞
HEK293A細胞(ヒト胎児腎細胞)は10%FCS、1%ペニシリン/ストレプトマイシンを含むDMEM培地を用いて37℃、5%CO2、湿度90%の条件下で培養した。マウスIgG抗体、ヒトプロインスリン、および、レプチンを恒常的に発現するHEK293A細胞株は、CMVプロモーターの下流にこれらのcDNAを挿入したコンストラクトをトランスフェクションした後、長期培養によって単一クローンを形成させた。その後、各クローンから目的タンパク質の発現をELISA法を用いて確認し、細胞株を樹立した。これらの細胞株も同様の条件で培養した。 HEK293A cells (human embryonic kidney cells) were cultured in DMEM medium containing 10% FCS and 1% penicillin/streptomycin under conditions of 37°C, 5% CO 2 and 90% humidity. A HEK293A cell line that constitutively expresses mouse IgG antibody, human proinsulin, and leptin was transfected with constructs that inserted these cDNAs downstream of the CMV promoter, followed by long-term culture to form single clones. . Thereafter, the expression of the target protein was confirmed from each clone using the ELISA method, and cell lines were established. These cell lines were also cultured under similar conditions.
HEK293A細胞(ヒト胎児腎細胞)は10%FCS、1%ペニシリン/ストレプトマイシンを含むDMEM培地を用いて37℃、5%CO2、湿度90%の条件下で培養した。マウスIgG抗体、ヒトプロインスリン、および、レプチンを恒常的に発現するHEK293A細胞株は、CMVプロモーターの下流にこれらのcDNAを挿入したコンストラクトをトランスフェクションした後、長期培養によって単一クローンを形成させた。その後、各クローンから目的タンパク質の発現をELISA法を用いて確認し、細胞株を樹立した。これらの細胞株も同様の条件で培養した。 HEK293A cells (human embryonic kidney cells) were cultured in DMEM medium containing 10% FCS and 1% penicillin/streptomycin under conditions of 37°C, 5% CO 2 and 90% humidity. A HEK293A cell line that constitutively expresses mouse IgG antibody, human proinsulin, and leptin was transfected with constructs that inserted these cDNAs downstream of the CMV promoter, followed by long-term culture to form single clones. . Thereafter, the expression of the target protein was confirmed from each clone using the ELISA method, and cell lines were established. These cell lines were also cultured under similar conditions.
トランスフェクション
HEK293A細胞を24ウェルプレートに1.0×105細胞/ウェルとなるように播種し、24時間培養した。特記しない限り、1ウェルあたり50ngの発現コンストラクト、1μl-Plus Reagent、1μl-Lipofectamine LTX を50μlのOpti-MEMで調整し、細胞を培養した24ウェルプレートの各ウェルに添加し、37℃で24時間培養することでトランスフェクションを行った。 Transfected HEK293A cells were seeded in a 24-well plate at 1.0×10 5 cells/well and cultured for 24 hours. Unless otherwise specified, 50 ng of expression construct, 1 μl-Plus Reagent, and 1 μl-Lipofectamine LTX per well were prepared in 50 μl of Opti-MEM, added to each well of a 24-well plate in which cells were cultured, and incubated at 37°C for 24 hours. Transfection was performed by culturing.
HEK293A細胞を24ウェルプレートに1.0×105細胞/ウェルとなるように播種し、24時間培養した。特記しない限り、1ウェルあたり50ngの発現コンストラクト、1μl-Plus Reagent、1μl-Lipofectamine LTX を50μlのOpti-MEMで調整し、細胞を培養した24ウェルプレートの各ウェルに添加し、37℃で24時間培養することでトランスフェクションを行った。 Transfected HEK293A cells were seeded in a 24-well plate at 1.0×10 5 cells/well and cultured for 24 hours. Unless otherwise specified, 50 ng of expression construct, 1 μl-Plus Reagent, and 1 μl-Lipofectamine LTX per well were prepared in 50 μl of Opti-MEM, added to each well of a 24-well plate in which cells were cultured, and incubated at 37°C for 24 hours. Transfection was performed by culturing.
ルシフェラーゼアッセイ
トランスフェクションから24時間後にウェルから培地を除去し、各濃度の化合物を添加した培地を加え、さらに24時間培養した。その後、培地を除去し、500μlPBSで洗浄した後、50μlの1x Glo Lysis buffer(Promega #E2661)で細胞を溶解した。この内、5μlをルシフェラーゼ活性測定に、5μlをタンパク質濃度測定に用いた。ルシフェラーゼ活性はセレンテラジンh(FUJIFILM #035-22991)の発光を検出することでRlucの発現量を定量し、また、タンパク質濃度はBCA法を用いて定量し、各ルシフェラーゼ活性はRluc/BCA値で算出した。なお、発光検出、BCA測定にはマルチラベルリーダー 2030 ARVOTM X(Perkin Elmer)を用いた。 Twenty-four hours after luciferase assay transfection, the medium was removed from the wells, medium supplemented with each concentration of compound was added, and cultured for an additional 24 hours. Thereafter, the medium was removed, washed with 500 µl of PBS, and then the cells were lysed with 50 µl of 1x Glo Lysis buffer (Promega #E2661). Of this, 5 μl was used for luciferase activity measurement, and 5 μl was used for protein concentration measurement. Luciferase activity was determined by detecting the luminescence of coelenterazine h (FUJIFILM #035-22991) to quantify the expression level of Rluc, protein concentration was quantified using the BCA method, and each luciferase activity was calculated as the Rluc/BCA value. bottom. A multi-label reader 2030 ARVO™ X (Perkin Elmer) was used for luminescence detection and BCA measurement.
トランスフェクションから24時間後にウェルから培地を除去し、各濃度の化合物を添加した培地を加え、さらに24時間培養した。その後、培地を除去し、500μlPBSで洗浄した後、50μlの1x Glo Lysis buffer(Promega #E2661)で細胞を溶解した。この内、5μlをルシフェラーゼ活性測定に、5μlをタンパク質濃度測定に用いた。ルシフェラーゼ活性はセレンテラジンh(FUJIFILM #035-22991)の発光を検出することでRlucの発現量を定量し、また、タンパク質濃度はBCA法を用いて定量し、各ルシフェラーゼ活性はRluc/BCA値で算出した。なお、発光検出、BCA測定にはマルチラベルリーダー 2030 ARVOTM X(Perkin Elmer)を用いた。 Twenty-four hours after luciferase assay transfection, the medium was removed from the wells, medium supplemented with each concentration of compound was added, and cultured for an additional 24 hours. Thereafter, the medium was removed, washed with 500 µl of PBS, and then the cells were lysed with 50 µl of 1x Glo Lysis buffer (Promega #E2661). Of this, 5 μl was used for luciferase activity measurement, and 5 μl was used for protein concentration measurement. Luciferase activity was determined by detecting the luminescence of coelenterazine h (FUJIFILM #035-22991) to quantify the expression level of Rluc, protein concentration was quantified using the BCA method, and each luciferase activity was calculated as the Rluc/BCA value. bottom. A multi-label reader 2030 ARVO™ X (Perkin Elmer) was used for luminescence detection and BCA measurement.
ウェスタンブロット
HEK293A細胞を6-ウェルプレートに5.0×105細胞/ウェルとなるように播種し、24時間培養した。その後、培地交換によって各化合物群を処理し、3時間後に細胞を2mlPBSで洗浄したのち、200μlRIPAバッファーを用いて溶解した。溶解した細胞を超音波処理を用いて破砕し、遠心(20,000g、15分、4℃)し、上清を回収し、タンパク質濃度を定量した。それらの溶解液を等量に合わせた後、1xSDSサンプルバッファーを加え、95℃で3分間熱処理した物をSDS-PAGEのサンプルとして用いた(1レーンあたり5μgのタンパク質)。SDS-PAGEのゲルはスーパーセップTMエース5-20%,17ウェル(Wako)のゲルを用い、ゲル1枚あたり
500V、40mA、35分間の条件下で電気泳動を行った。その後、ブロッキング装置(ATTO)を用いてゲル1枚あたり500V、100mA、60分間の条件でPVDFメンブレンに転写した。転写後のメンブレンをブロッキング溶液(3%BSA/TBS-T)を用いて室温で30分間浸透し、一次抗体の反応は、ブロッキング溶液に一次抗体(Phospho-PKC Substrate Motif [(R/K)XpSX(R/K)] MultiMabTM Rabbit mAb mix, Cell Signaling Technology #6967))を3000倍希釈の条件下、4℃で一晩で行った。その後、TBS-Tを用いてメンブレンを3回洗浄し、二次抗体反応を、TBS-T中にHRP標識二次抗体(Anti-Rabbit IgG, HRP-Linked Whole Ab Donkey, Cytiva #NA934-1ML)を5000倍希釈の条件下、室温で3時間行った。その後、TBSTを用いてメンブレンを3回洗浄し、Chemi-Lumi One(nacalai #07880-70)と ImageQuant LAS4010 を用いて検出を行った。 Western blot HEK293A cells were seeded in a 6-well plate at 5.0×10 5 cells/well and cultured for 24 hours. Thereafter, each compound group was treated by medium exchange, and after 3 hours the cells were washed with 2 ml of PBS and lysed with 200 μl of RIPA buffer. Lysed cells were disrupted using sonication, centrifuged (20,000 g, 15 min, 4° C.), the supernatant collected and protein concentration quantified. After equal amounts of these lysates were combined, 1×SDS sample buffer was added, and the mixture was heat-treated at 95° C. for 3 minutes and used as a sample for SDS-PAGE (5 μg of protein per lane). SuperSep™ Ace 5-20%, 17-well (Wako) gels were used as SDS-PAGE gels, and electrophoresis was performed under the conditions of 500 V, 40 mA, and 35 minutes per gel. Thereafter, using a blocking device (ATTO), each gel was transferred to a PVDF membrane under conditions of 500 V, 100 mA, and 60 minutes. After transfer, the membrane was permeated with a blocking solution (3% BSA/TBS-T) for 30 minutes at room temperature. (R/K)] MultiMabTM Rabbit mAb mix, Cell Signaling Technology #6967)) was performed overnight at 4°C under conditions of 3000-fold dilution. After that, the membrane was washed three times with TBS-T, and the secondary antibody reaction was performed by adding HRP-labeled secondary antibody (Anti-Rabbit IgG, HRP-Linked Whole Ab Donkey, Cytiva #NA934-1ML) in TBS-T. was performed at room temperature for 3 hours under conditions of 5000-fold dilution. After that, the membrane was washed three times with TBST and detected using Chemi-Lumi One (nacalai #07880-70) and ImageQuant LAS4010.
HEK293A細胞を6-ウェルプレートに5.0×105細胞/ウェルとなるように播種し、24時間培養した。その後、培地交換によって各化合物群を処理し、3時間後に細胞を2mlPBSで洗浄したのち、200μlRIPAバッファーを用いて溶解した。溶解した細胞を超音波処理を用いて破砕し、遠心(20,000g、15分、4℃)し、上清を回収し、タンパク質濃度を定量した。それらの溶解液を等量に合わせた後、1xSDSサンプルバッファーを加え、95℃で3分間熱処理した物をSDS-PAGEのサンプルとして用いた(1レーンあたり5μgのタンパク質)。SDS-PAGEのゲルはスーパーセップTMエース5-20%,17ウェル(Wako)のゲルを用い、ゲル1枚あたり
500V、40mA、35分間の条件下で電気泳動を行った。その後、ブロッキング装置(ATTO)を用いてゲル1枚あたり500V、100mA、60分間の条件でPVDFメンブレンに転写した。転写後のメンブレンをブロッキング溶液(3%BSA/TBS-T)を用いて室温で30分間浸透し、一次抗体の反応は、ブロッキング溶液に一次抗体(Phospho-PKC Substrate Motif [(R/K)XpSX(R/K)] MultiMabTM Rabbit mAb mix, Cell Signaling Technology #6967))を3000倍希釈の条件下、4℃で一晩で行った。その後、TBS-Tを用いてメンブレンを3回洗浄し、二次抗体反応を、TBS-T中にHRP標識二次抗体(Anti-Rabbit IgG, HRP-Linked Whole Ab Donkey, Cytiva #NA934-1ML)を5000倍希釈の条件下、室温で3時間行った。その後、TBSTを用いてメンブレンを3回洗浄し、Chemi-Lumi One(nacalai #07880-70)と ImageQuant LAS4010 を用いて検出を行った。 Western blot HEK293A cells were seeded in a 6-well plate at 5.0×10 5 cells/well and cultured for 24 hours. Thereafter, each compound group was treated by medium exchange, and after 3 hours the cells were washed with 2 ml of PBS and lysed with 200 μl of RIPA buffer. Lysed cells were disrupted using sonication, centrifuged (20,000 g, 15 min, 4° C.), the supernatant collected and protein concentration quantified. After equal amounts of these lysates were combined, 1×SDS sample buffer was added, and the mixture was heat-treated at 95° C. for 3 minutes and used as a sample for SDS-PAGE (5 μg of protein per lane). SuperSep™ Ace 5-20%, 17-well (Wako) gels were used as SDS-PAGE gels, and electrophoresis was performed under the conditions of 500 V, 40 mA, and 35 minutes per gel. Thereafter, using a blocking device (ATTO), each gel was transferred to a PVDF membrane under conditions of 500 V, 100 mA, and 60 minutes. After transfer, the membrane was permeated with a blocking solution (3% BSA/TBS-T) for 30 minutes at room temperature. (R/K)] MultiMabTM Rabbit mAb mix, Cell Signaling Technology #6967)) was performed overnight at 4°C under conditions of 3000-fold dilution. After that, the membrane was washed three times with TBS-T, and the secondary antibody reaction was performed by adding HRP-labeled secondary antibody (Anti-Rabbit IgG, HRP-Linked Whole Ab Donkey, Cytiva #NA934-1ML) in TBS-T. was performed at room temperature for 3 hours under conditions of 5000-fold dilution. After that, the membrane was washed three times with TBST and detected using Chemi-Lumi One (nacalai #07880-70) and ImageQuant LAS4010.
ELISA法
各分泌タンパク質発現細胞を24ウェルプレートに1.0×104細胞/ウェルとなるように播種し、24時間培養した。その後、1ml/ウェルの条件で培地交換によって各化合物群を処理し、24時間ごとに培地を120μl回収、冷蔵保存した。その後、各ELISAキット(マウスIgG, Betyl Lab #E99-131, ヒトプロインスリン: Mercodia #10-1118-01, ヒトレプチン: Proteintech #KE00095)を用いて培地中の目的タンパク質の濃度を測定した。測定にはマルチラベルリーダー2030 ARVOTM X(Perkin Elmer)を用いた。 ELISA method Cells expressing each secretory protein were seeded in a 24-well plate at 1.0×10 4 cells/well and cultured for 24 hours. Thereafter, each compound group was treated by medium exchange under the condition of 1 ml/well, and 120 μl of medium was collected every 24 hours and stored in a refrigerator. After that, each ELISA kit (mouse IgG, Betyl Lab #E99-131, human proinsulin: Mercodia #10-1118-01, human leptin: Proteintech #KE00095) was used to measure the concentration of the target protein in the medium. A multi-label reader 2030 ARVO™ X (Perkin Elmer) was used for the measurement.
各分泌タンパク質発現細胞を24ウェルプレートに1.0×104細胞/ウェルとなるように播種し、24時間培養した。その後、1ml/ウェルの条件で培地交換によって各化合物群を処理し、24時間ごとに培地を120μl回収、冷蔵保存した。その後、各ELISAキット(マウスIgG, Betyl Lab #E99-131, ヒトプロインスリン: Mercodia #10-1118-01, ヒトレプチン: Proteintech #KE00095)を用いて培地中の目的タンパク質の濃度を測定した。測定にはマルチラベルリーダー2030 ARVOTM X(Perkin Elmer)を用いた。 ELISA method Cells expressing each secretory protein were seeded in a 24-well plate at 1.0×10 4 cells/well and cultured for 24 hours. Thereafter, each compound group was treated by medium exchange under the condition of 1 ml/well, and 120 μl of medium was collected every 24 hours and stored in a refrigerator. After that, each ELISA kit (mouse IgG, Betyl Lab #E99-131, human proinsulin: Mercodia #10-1118-01, human leptin: Proteintech #KE00095) was used to measure the concentration of the target protein in the medium. A multi-label reader 2030 ARVO™ X (Perkin Elmer) was used for the measurement.
試験1:CMVプロモーターの転写活性を増強する化合物の同定
HEK293T細胞にCMVプロモーターの下流にルシフェラーゼ遺伝子を挿入したプラスミドを24-ウェルプレートの1ウェルあたり20ngでトランスフェクションし、24時間後に30μg/mlの植物エキスを含んだ培地に培地交換した。その後、24時間後にルシフェラーゼアッセイを用いて、CMVプロモーター活性を測定した。本研究では約1000種類の植物エキスを用いた。独自の植物(生薬原料)エキスライブラリーから、CMVプロモーターの転写活性を劇的に増強する5種類の植物エキスおよびその活性を担う化合物(下記の8種類)を同定した。
Test 1: Identification of compounds that enhance the transcriptional activity of the CMV promoter HEK293T cells were transfected with 20 ng per well of a 24-well plate with a plasmid having a luciferase gene inserted downstream of the CMV promoter. The medium was replaced with a medium containing a plant extract. CMV promoter activity was then measured 24 hours later using a luciferase assay. About 1000 types of plant extracts were used in this study. Five types of plant extracts that dramatically enhance the transcriptional activity of the CMV promoter and compounds responsible for the activity (the following eight types) were identified from a proprietary plant (raw drug material) extract library.
HEK293T細胞にCMVプロモーターの下流にルシフェラーゼ遺伝子を挿入したプラスミドを24-ウェルプレートの1ウェルあたり20ngでトランスフェクションし、24時間後に30μg/mlの植物エキスを含んだ培地に培地交換した。その後、24時間後にルシフェラーゼアッセイを用いて、CMVプロモーター活性を測定した。本研究では約1000種類の植物エキスを用いた。独自の植物(生薬原料)エキスライブラリーから、CMVプロモーターの転写活性を劇的に増強する5種類の植物エキスおよびその活性を担う化合物(下記の8種類)を同定した。
試験2:化合物の活性はプロテインキナーゼC(PKC)の阻害剤により阻害される
pRL-CMVを恒常的に発現するHEK293A細胞を、図1に示す濃度で化合物Xまたは化合物#1~#7を含む培地中、PKC阻害剤の存在下または非存在下で24時間培養し、ルシフェラーゼ活性を測定した。結果を図1に示す。CMVプロモーターの転写活性は、化合物の存在下で増強され、その増強はPKC阻害剤により阻害された。 Test 2: Activity of Compounds is Inhibited by Inhibitors of Protein Kinase C (PKC) HEK293A cells constitutively expressing pRL-CMV were treated with Compound X or Compounds #1-#7 at the concentrations indicated in FIG. The cells were cultured in the medium in the presence or absence of PKC inhibitors for 24 hours, and luciferase activity was measured. The results are shown in FIG. Transcriptional activity of the CMV promoter was enhanced in the presence of compounds, and the enhancement was inhibited by PKC inhibitors.
pRL-CMVを恒常的に発現するHEK293A細胞を、図1に示す濃度で化合物Xまたは化合物#1~#7を含む培地中、PKC阻害剤の存在下または非存在下で24時間培養し、ルシフェラーゼ活性を測定した。結果を図1に示す。CMVプロモーターの転写活性は、化合物の存在下で増強され、その増強はPKC阻害剤により阻害された。 Test 2: Activity of Compounds is Inhibited by Inhibitors of Protein Kinase C (PKC) HEK293A cells constitutively expressing pRL-CMV were treated with Compound X or Compounds #1-#7 at the concentrations indicated in FIG. The cells were cultured in the medium in the presence or absence of PKC inhibitors for 24 hours, and luciferase activity was measured. The results are shown in FIG. Transcriptional activity of the CMV promoter was enhanced in the presence of compounds, and the enhancement was inhibited by PKC inhibitors.
pRL-CMVを恒常的に発現するHEK293A細胞を、化合物X(100ng/ml)を添加した培地中で24時間培養し、ルシフェラーゼ活性を経時的に測定した。結果を図2に示す。化合物XによるCMVプロモーターの転写活性増強は、化合物X添加の1時間後から観察された。
HEK293A cells that constitutively express pRL-CMV were cultured in medium supplemented with compound X (100 ng/ml) for 24 hours, and luciferase activity was measured over time. The results are shown in FIG. Enhancement of transcriptional activity of the CMV promoter by Compound X was observed from 1 hour after addition of Compound X.
試験3:CMVプロモーターの活性は既知のPKC活性化剤によっても増強される
pRL-CMVを恒常的に発現するHEK293A細胞を、図3に示す濃度で化合物XまたはPKC活性化剤#1~#4を含む培地中、PKC阻害剤の存在下または非存在下で24時間培養し、ルシフェラーゼ活性を測定した。結果を図3に示す。CMVプロモーターの転写活性は、既知のPKC活性化剤によっても増強された。 Test 3: CMV promoter activity is also enhanced by known PKC activators. was cultured for 24 hours in the presence or absence of a PKC inhibitor in a medium containing the luciferase activity was measured. The results are shown in FIG. CMV promoter transcriptional activity was also enhanced by known PKC activators.
pRL-CMVを恒常的に発現するHEK293A細胞を、図3に示す濃度で化合物XまたはPKC活性化剤#1~#4を含む培地中、PKC阻害剤の存在下または非存在下で24時間培養し、ルシフェラーゼ活性を測定した。結果を図3に示す。CMVプロモーターの転写活性は、既知のPKC活性化剤によっても増強された。 Test 3: CMV promoter activity is also enhanced by known PKC activators. was cultured for 24 hours in the presence or absence of a PKC inhibitor in a medium containing the luciferase activity was measured. The results are shown in FIG. CMV promoter transcriptional activity was also enhanced by known PKC activators.
化合物X、化合物#1~#7およびPKC活性化剤#1~#4は、テルペンに属し、ジテルペンと総称される化合物である。骨格により以下の3種類に分類される。
Compound X, compounds #1-#7 and PKC activators #1-#4 belong to terpenes and are compounds collectively called diterpenes. It is classified into the following three types according to the skeleton.
試験4:CMVプロモーターの活性はジテルペン以外のPKC活性化剤によっても増強される
pRL-CMVを恒常的に発現するHEK293A細胞を、図4に示す濃度で化合物XまたはPKC活性化剤#5~#6を含む培地中、PKC阻害剤(Ro-318425)の存在下または非存在下で24時間培養し、ルシフェラーゼ活性を測定した。結果を図4に示す。CMVプロモーターの転写活性は、ジテルペン以外のPKC活性化剤によっても増強された。 Test 4: CMV promoter activity is also enhanced by PKC activators other than diterpenes. The cells were cultured in medium containing 6 in the presence or absence of a PKC inhibitor (Ro-318425) for 24 hours, and luciferase activity was measured. The results are shown in FIG. CMV promoter transcriptional activity was also enhanced by PKC activators other than diterpenes.
pRL-CMVを恒常的に発現するHEK293A細胞を、図4に示す濃度で化合物XまたはPKC活性化剤#5~#6を含む培地中、PKC阻害剤(Ro-318425)の存在下または非存在下で24時間培養し、ルシフェラーゼ活性を測定した。結果を図4に示す。CMVプロモーターの転写活性は、ジテルペン以外のPKC活性化剤によっても増強された。 Test 4: CMV promoter activity is also enhanced by PKC activators other than diterpenes. The cells were cultured in medium containing 6 in the presence or absence of a PKC inhibitor (Ro-318425) for 24 hours, and luciferase activity was measured. The results are shown in FIG. CMV promoter transcriptional activity was also enhanced by PKC activators other than diterpenes.
試験5:化合物はCAGプロモーターおよびEF1プロモーターの活性も増強する
HEK293A細胞に、pCAG-Rluc、もしくはpEF1-Rlucをトランスフェクションし、図5~7に示す濃度で化合物X、化合物#1~#7またはPKC活性化剤#1~#6を含む培地中で培養し、pCAGGSベクター由来のCAGプロモーターおよびpEFBOSベクター由来のEF1プロモーターの転写活性を測定した。結果を図5~7に示す。CAGプロモーターおよびEF1プロモーターの転写活性は、化合物またはPKC活性化剤の存在下で増強されたが、その増強はPKC阻害剤により阻害された。これらのプロモーターは、SP1、CEBP、AP1、NF-κB、YY1などの転写因子の結合部位を有する。従って、これらの化合物およびPKC活性化剤は、共通の転写因子を介してこれらのプロモーターを活性化することが示唆される。 Test 5: Compounds also enhance CAG and EF1 promoter activity The cells were cultured in media containingPKC activators # 1 to #6, and the transcriptional activities of the pCAGGS vector-derived CAG promoter and the pEFBOS vector-derived EF1 promoter were measured. The results are shown in Figures 5-7. CAG and EF1 promoter transcriptional activities were enhanced in the presence of compounds or PKC activators, but the enhancement was inhibited by PKC inhibitors. These promoters have binding sites for transcription factors such as SP1, CEBP, AP1, NF-κB, YY1. Therefore, it is suggested that these compounds and PKC activators activate these promoters through common transcription factors.
HEK293A細胞に、pCAG-Rluc、もしくはpEF1-Rlucをトランスフェクションし、図5~7に示す濃度で化合物X、化合物#1~#7またはPKC活性化剤#1~#6を含む培地中で培養し、pCAGGSベクター由来のCAGプロモーターおよびpEFBOSベクター由来のEF1プロモーターの転写活性を測定した。結果を図5~7に示す。CAGプロモーターおよびEF1プロモーターの転写活性は、化合物またはPKC活性化剤の存在下で増強されたが、その増強はPKC阻害剤により阻害された。これらのプロモーターは、SP1、CEBP、AP1、NF-κB、YY1などの転写因子の結合部位を有する。従って、これらの化合物およびPKC活性化剤は、共通の転写因子を介してこれらのプロモーターを活性化することが示唆される。 Test 5: Compounds also enhance CAG and EF1 promoter activity The cells were cultured in media containing
試験6:化合物群はPKCを活性化する
HEK293A細胞を、試験2~5と同様の濃度で化合物X、化合物#1~#7、PKC活性化剤#1~#6、化合物SCまたは酪酸ナトリウム(SB)を含む培地中、PKC阻害剤(3μM)の存在下または非存在下で3時間培養した。細胞を溶解し、PKCによってリン酸化されたタンパク質を特異的に認識する抗体を用いてウェスタンブロットを行った。結果を図8に示す。化合物X、化合物#1~#7およびPKC活性化剤#1~#6は、PKCを活性化した。化合物SCおよび酪酸ナトリウムによるPKC活性化は確認されなかった。 Test 6: Compounds Activate PKC HEK293A cells were treated with Compound X, Compounds #1-#7, PKC activators #1-#6, Compound SC or sodium butyrate ( SB) in the presence or absence of a PKC inhibitor (3 μM) for 3 hours. Cells were lysed and Western blots were performed using an antibody that specifically recognizes proteins phosphorylated by PKC. The results are shown in FIG. Compound X, compounds #1-#7 and PKC activators #1-#6 activated PKC. PKC activation by Compound SC and sodium butyrate was not confirmed.
HEK293A細胞を、試験2~5と同様の濃度で化合物X、化合物#1~#7、PKC活性化剤#1~#6、化合物SCまたは酪酸ナトリウム(SB)を含む培地中、PKC阻害剤(3μM)の存在下または非存在下で3時間培養した。細胞を溶解し、PKCによってリン酸化されたタンパク質を特異的に認識する抗体を用いてウェスタンブロットを行った。結果を図8に示す。化合物X、化合物#1~#7およびPKC活性化剤#1~#6は、PKCを活性化した。化合物SCおよび酪酸ナトリウムによるPKC活性化は確認されなかった。 Test 6: Compounds Activate PKC HEK293A cells were treated with Compound X, Compounds #1-#7, PKC activators #1-#6, Compound SC or sodium butyrate ( SB) in the presence or absence of a PKC inhibitor (3 μM) for 3 hours. Cells were lysed and Western blots were performed using an antibody that specifically recognizes proteins phosphorylated by PKC. The results are shown in FIG. Compound X, compounds #1-#7 and PKC activators #1-#6 activated PKC. PKC activation by Compound SC and sodium butyrate was not confirmed.
試験7:新たなCMVプロモーター活性化剤の同定
pRL-CMVを恒常的に発現するHEK293A細胞を、図9に示す濃度で化合物Xおよび/または化合物SCを含む培地中、PKC阻害剤の存在下または非存在下で24時間培養し、ルシフェラーゼ活性を測定した。結果を図9に示す。CMVプロモーターの転写活性は、化合物SCによっても増強されたが、この増強はPKC阻害剤により阻害されなかった。化合物Xと化合物SCを併用すると転写活性はさらに増強された。 Test 7: Identification of New CMV Promoter Activators HEK293A cells constitutively expressing pRL-CMV were incubated in medium containing compound X and/or compound SC at the concentrations shown in FIG. After culturing for 24 hours in the absence, luciferase activity was measured. The results are shown in FIG. CMV promoter transcriptional activity was also enhanced by compound SC, but this enhancement was not inhibited by PKC inhibitors. Transcriptional activity was further enhanced when compound X and compound SC were used in combination.
pRL-CMVを恒常的に発現するHEK293A細胞を、図9に示す濃度で化合物Xおよび/または化合物SCを含む培地中、PKC阻害剤の存在下または非存在下で24時間培養し、ルシフェラーゼ活性を測定した。結果を図9に示す。CMVプロモーターの転写活性は、化合物SCによっても増強されたが、この増強はPKC阻害剤により阻害されなかった。化合物Xと化合物SCを併用すると転写活性はさらに増強された。 Test 7: Identification of New CMV Promoter Activators HEK293A cells constitutively expressing pRL-CMV were incubated in medium containing compound X and/or compound SC at the concentrations shown in FIG. After culturing for 24 hours in the absence, luciferase activity was measured. The results are shown in FIG. CMV promoter transcriptional activity was also enhanced by compound SC, but this enhancement was not inhibited by PKC inhibitors. Transcriptional activity was further enhanced when compound X and compound SC were used in combination.
試験8:PKC活性化剤、化合物SCおよびヒストンデアセチラーゼ阻害剤の併用によりCMVプロモーターの転写活性はさらに増強される
pRL-CMVを恒常的に発現するHEK293A細胞を、図10に示す濃度で化合物X、化合物SCおよび/または酪酸ナトリウム(SB)を含む培地中で24時間培養し、ルシフェラーゼ活性を測定した。結果を図10に示す。CMVプロモーターの転写活性は、化合物X、化合物SCおよび酪酸ナトリウムを併用すると最も高かった。同様の結果が化合物#1~#7およびPKC活性化剤#1~#6でも得られた(図11および12)。酪酸ナトリウムの代わりにヒストンデアセチラーゼ阻害剤のバルプロ酸、トリコスタチンA(TSA)、ボリノスタット(SAHA)、アピシジン、エンチノスタット(MS-275)またはタセジナリン(CI994)を用いても、同様の結果が得られた(図13)。 Test 8: Combined use of PKC activator, compound SC and histone deacetylase inhibitor further enhances transcriptional activity of CMV promoter. X, compound SC and/or sodium butyrate (SB) were cultured for 24 hours and luciferase activity was measured. The results are shown in FIG. Transcriptional activity of the CMV promoter was highest when compound X, compound SC and sodium butyrate were combined. Similar results were obtained with compounds #1-#7 and PKC activators #1-#6 (FIGS. 11 and 12). Similar results were obtained using the histone deacetylase inhibitors valproic acid, trichostatin A (TSA), vorinostat (SAHA), apicidin, entinostat (MS-275) or tacedinaline (CI994) instead of sodium butyrate. was obtained (FIG. 13).
pRL-CMVを恒常的に発現するHEK293A細胞を、図10に示す濃度で化合物X、化合物SCおよび/または酪酸ナトリウム(SB)を含む培地中で24時間培養し、ルシフェラーゼ活性を測定した。結果を図10に示す。CMVプロモーターの転写活性は、化合物X、化合物SCおよび酪酸ナトリウムを併用すると最も高かった。同様の結果が化合物#1~#7およびPKC活性化剤#1~#6でも得られた(図11および12)。酪酸ナトリウムの代わりにヒストンデアセチラーゼ阻害剤のバルプロ酸、トリコスタチンA(TSA)、ボリノスタット(SAHA)、アピシジン、エンチノスタット(MS-275)またはタセジナリン(CI994)を用いても、同様の結果が得られた(図13)。 Test 8: Combined use of PKC activator, compound SC and histone deacetylase inhibitor further enhances transcriptional activity of CMV promoter. X, compound SC and/or sodium butyrate (SB) were cultured for 24 hours and luciferase activity was measured. The results are shown in FIG. Transcriptional activity of the CMV promoter was highest when compound X, compound SC and sodium butyrate were combined. Similar results were obtained with compounds #1-#7 and PKC activators #1-#6 (FIGS. 11 and 12). Similar results were obtained using the histone deacetylase inhibitors valproic acid, trichostatin A (TSA), vorinostat (SAHA), apicidin, entinostat (MS-275) or tacedinaline (CI994) instead of sodium butyrate. was obtained (FIG. 13).
試験9:PKC活性化剤によるタンパク質生産の増加
pcDNAベクターのCMVプロモーターの下流にマウスIgG抗体重鎖、マウスIgG抗体軽鎖、ヒトプロインスリンまたはヒトレプチンのcDNAを挿入し、発現コンストラクトを作製した。マウスIgG抗体およびヒトレプチンのコンストラクトをそれぞれHEK293A細胞にトランスフェクションした。ヒトプロインスリンのコンストラクトをHEK293A細胞にトランスフェクションし、ヒトプロインスリンを恒常的に発現するHEK293A細胞を作製した。図14~17に示す濃度で化合物X、化合物#1~#7、PKC活性化剤#1~#6を含む培地中、化合物SCまたは化合物SC+酪酸ナトリウム(SB)の存在下または非存在下で細胞を24時間培養し、培地中の各タンパク質の濃度をELISA法により測定した。結果を図14~17に示す。さらに、化合物Xについて、マウスIgG抗体重鎖および軽鎖、ヒトプロインスリンまたはヒトレプチンを恒常的に発現するHEK293A細胞を用い、酪酸ナトリウム(SB)の存在下または非存在下で、24時間毎に4日間にわたりタンパク質濃度を測定した。結果を図18に示す。IgG抗体、プロインスリンおよびレプチンの生産はこれらの化合物により増強された。IgG抗体およびプロインスリンの生産は、これらの化合物と化合物SCおよび酪酸ナトリウムを併用するとさらに増強された。レプチンの生産は、これらの化合物と化合物SCを併用した場合に最も増強され、SBの存在下では減少したが、いずれの物質も含まない培地で培養した場合よりは多かった。 Test 9: Increased Protein Production by PKC Activator Expression constructs were prepared by inserting cDNAs of mouse IgG antibody heavy chain, mouse IgG antibody light chain, human proinsulin or human leptin downstream of the CMV promoter of a pcDNA vector. Mouse IgG antibody and human leptin constructs were transfected into HEK293A cells, respectively. The human proinsulin construct was transfected into HEK293A cells to generate HEK293A cells that constitutively express human proinsulin. In the presence or absence of Compound SC or Compound SC plus sodium butyrate (SB) in media containing Compound X, Compounds #1-#7, PKC activators #1-#6 at the concentrations shown in FIGS. Cells were cultured for 24 hours and the concentration of each protein in the medium was measured by ELISA method. The results are shown in Figures 14-17. In addition, for compound X, HEK293A cells constitutively expressing mouse IgG antibody heavy and light chains, human proinsulin or human leptin were used in the presence or absence of sodium butyrate (SB) every 24 hours. Protein concentrations were measured over the course of the day. The results are shown in FIG. Production of IgG antibodies, proinsulin and leptin was enhanced by these compounds. IgG antibody and proinsulin production were further enhanced when these compounds were combined with compound SC and sodium butyrate. Leptin production was most enhanced when these compounds were combined with compound SC, and decreased in the presence of SB, but more than when cultured in medium without either substance.
pcDNAベクターのCMVプロモーターの下流にマウスIgG抗体重鎖、マウスIgG抗体軽鎖、ヒトプロインスリンまたはヒトレプチンのcDNAを挿入し、発現コンストラクトを作製した。マウスIgG抗体およびヒトレプチンのコンストラクトをそれぞれHEK293A細胞にトランスフェクションした。ヒトプロインスリンのコンストラクトをHEK293A細胞にトランスフェクションし、ヒトプロインスリンを恒常的に発現するHEK293A細胞を作製した。図14~17に示す濃度で化合物X、化合物#1~#7、PKC活性化剤#1~#6を含む培地中、化合物SCまたは化合物SC+酪酸ナトリウム(SB)の存在下または非存在下で細胞を24時間培養し、培地中の各タンパク質の濃度をELISA法により測定した。結果を図14~17に示す。さらに、化合物Xについて、マウスIgG抗体重鎖および軽鎖、ヒトプロインスリンまたはヒトレプチンを恒常的に発現するHEK293A細胞を用い、酪酸ナトリウム(SB)の存在下または非存在下で、24時間毎に4日間にわたりタンパク質濃度を測定した。結果を図18に示す。IgG抗体、プロインスリンおよびレプチンの生産はこれらの化合物により増強された。IgG抗体およびプロインスリンの生産は、これらの化合物と化合物SCおよび酪酸ナトリウムを併用するとさらに増強された。レプチンの生産は、これらの化合物と化合物SCを併用した場合に最も増強され、SBの存在下では減少したが、いずれの物質も含まない培地で培養した場合よりは多かった。 Test 9: Increased Protein Production by PKC Activator Expression constructs were prepared by inserting cDNAs of mouse IgG antibody heavy chain, mouse IgG antibody light chain, human proinsulin or human leptin downstream of the CMV promoter of a pcDNA vector. Mouse IgG antibody and human leptin constructs were transfected into HEK293A cells, respectively. The human proinsulin construct was transfected into HEK293A cells to generate HEK293A cells that constitutively express human proinsulin. In the presence or absence of Compound SC or Compound SC plus sodium butyrate (SB) in media containing Compound X, Compounds #1-#7, PKC activators #1-#6 at the concentrations shown in FIGS. Cells were cultured for 24 hours and the concentration of each protein in the medium was measured by ELISA method. The results are shown in Figures 14-17. In addition, for compound X, HEK293A cells constitutively expressing mouse IgG antibody heavy and light chains, human proinsulin or human leptin were used in the presence or absence of sodium butyrate (SB) every 24 hours. Protein concentrations were measured over the course of the day. The results are shown in FIG. Production of IgG antibodies, proinsulin and leptin was enhanced by these compounds. IgG antibody and proinsulin production were further enhanced when these compounds were combined with compound SC and sodium butyrate. Leptin production was most enhanced when these compounds were combined with compound SC, and decreased in the presence of SB, but more than when cultured in medium without either substance.
試験10:遺伝子工学的PKC活性化によるCMVプロモーターの活性化
遺伝子工学的にPKCを活性化して、CMVプロモーターの転写活性を調べた。CMVプロモーターの下流に、タンパク質のcDNA(XXX)、IRESおよび活性化型PKCのcDNAを連結した発現コンストラクトを使用した。活性化型PKCとして、ヒトPKCδの334-695アミノ酸領域(PKCδ-CA)、ヒトPKCαの326-672アミノ酸領域の489番目のメチオニンをバリンに変えた恒常的活性化体(PKCαCA-M489V)を使用した。またXXXにはRluc、マウスIgG抗体(重鎖、軽鎖)、およびヒトレプチン遺伝子を挿入した。これらのコンストラクトの模式図を図19に示す。 Test 10: Activation of CMV promoter by genetically engineered PKC activation PKC was genetically engineered to examine the transcriptional activity of the CMV promoter. An expression construct was used in which the cDNA for the protein (XXX), the IRES and the cDNA for activated PKC were linked downstream of the CMV promoter. As the activated PKC, the 334-695 amino acid region of human PKCδ (PKCδ-CA) and the constitutively activated form (PKCαCA-M489V) in which the 489th methionine in the 326-672 amino acid region of human PKCα was changed to valine were used. bottom. In XXX, Rluc, mouse IgG antibody (heavy chain, light chain), and human leptin gene were inserted. Schematic representations of these constructs are shown in FIG.
遺伝子工学的にPKCを活性化して、CMVプロモーターの転写活性を調べた。CMVプロモーターの下流に、タンパク質のcDNA(XXX)、IRESおよび活性化型PKCのcDNAを連結した発現コンストラクトを使用した。活性化型PKCとして、ヒトPKCδの334-695アミノ酸領域(PKCδ-CA)、ヒトPKCαの326-672アミノ酸領域の489番目のメチオニンをバリンに変えた恒常的活性化体(PKCαCA-M489V)を使用した。またXXXにはRluc、マウスIgG抗体(重鎖、軽鎖)、およびヒトレプチン遺伝子を挿入した。これらのコンストラクトの模式図を図19に示す。 Test 10: Activation of CMV promoter by genetically engineered PKC activation PKC was genetically engineered to examine the transcriptional activity of the CMV promoter. An expression construct was used in which the cDNA for the protein (XXX), the IRES and the cDNA for activated PKC were linked downstream of the CMV promoter. As the activated PKC, the 334-695 amino acid region of human PKCδ (PKCδ-CA) and the constitutively activated form (PKCαCA-M489V) in which the 489th methionine in the 326-672 amino acid region of human PKCα was changed to valine were used. bottom. In XXX, Rluc, mouse IgG antibody (heavy chain, light chain), and human leptin gene were inserted. Schematic representations of these constructs are shown in FIG.
pcDNAベクターのCMVプロモーターの下流に、RlucのcDNA、IRES、および、PKCδ-CAまたはPKCαCA-M489VのcDNAを挿入し、発現コンストラクトを作製した。PKCを欠く対照のコンストラクトも作製した。これらのコンストラクトを6.25~200ng/ウェルの濃度でHEK293A細胞にトランスフェクションし、24時間培養し、ルシフェラーゼ活性を測定した。結果を図20に示す。PKC群では、ルシフェラーゼ活性が高かった。この結果は、CMVプロモーターの転写活性がポジティブフィードバックにより増強されることを示す。IRESの代わりに2A自己切断ペプチド配列であるP2Aペプチド配列を用いた発現コンストラクトでも、同様の結果が得られた(図21)。
An expression construct was prepared by inserting Rluc cDNA, IRES, and PKCδ-CA or PKCαCA-M489V cDNA downstream of the CMV promoter in a pcDNA vector. A control construct lacking PKC was also made. HEK293A cells were transfected with these constructs at concentrations of 6.25-200 ng/well, cultured for 24 hours, and luciferase activity was measured. The results are shown in FIG. The PKC group had higher luciferase activity. This result indicates that the transcriptional activity of the CMV promoter is enhanced by positive feedback. Similar results were obtained with an expression construct using the P2A peptide sequence, a 2A self-cleaving peptide sequence, instead of the IRES (Fig. 21).
pcDNAベクターのCMVプロモーターの下流に、マウスIgG抗体重鎖または軽鎖のcDNA、IRES、および、PKCδ-CAまたはPKCαCA-M489VのcDNAを挿入し、発現コンストラクトを作製した。PKCを欠く対照のコンストラクトも作製した。IgG抗体重鎖および軽鎖のコンストラクトを組み合わせて、100ng/ウェルの濃度でHEK293A細胞にトランスフェクションし、24時間培養し、培地中のIgG抗体の濃度をELISA法により24時間毎に4日間にわたって測定した。結果を図22に示す。PKC群では、IgG抗体の生産が増強された。この結果は、遺伝子工学的PKC活性化により、CMVプロモーターを活性化し、タンパク質生産を増強できることを示す。
A mouse IgG antibody heavy chain or light chain cDNA, IRES, and PKCδ-CA or PKCαCA-M489V cDNA were inserted downstream of the CMV promoter in a pcDNA vector to prepare an expression construct. A control construct lacking PKC was also made. HEK293A cells were transfected with combined IgG antibody heavy and light chain constructs at a concentration of 100 ng/well, cultured for 24 hours, and the concentration of IgG antibodies in the medium was measured by ELISA every 24 hours for 4 days. bottom. The results are shown in FIG. In the PKC group, IgG antibody production was enhanced. This result indicates that genetically engineered PKC activation can activate the CMV promoter and enhance protein production.
pcDNAベクターのCMVプロモーターの下流に、ヒトレプチンのcDNA、IRES、および、PKCαCA-M489VのcDNAを挿入し、発現コンストラクトを作製した。PKCを欠く対照のコンストラクトも作製した。これらのコンストラクトを100ng/ウェルの濃度でHEK293A細胞にトランスフェクションし、24時間培養し、培地中のレプチンの濃度をELISA法により4日間にわたって測定した。結果を図23に示す。PKC群では、レプチンの生産が増強された。この結果は、遺伝子工学的PKC活性化により、CMVプロモーターを活性化し、タンパク質生産を増強できることを示す。
An expression construct was prepared by inserting human leptin cDNA, IRES, and PKCαCA-M489V cDNA downstream of the CMV promoter of the pcDNA vector. A control construct lacking PKC was also made. HEK293A cells were transfected with these constructs at a concentration of 100 ng/well, cultured for 24 hours, and the concentration of leptin in the medium was measured by ELISA over 4 days. The results are shown in FIG. In the PKC group, leptin production was enhanced. This result indicates that genetically engineered PKC activation can activate the CMV promoter and enhance protein production.
試験11:化合物SCの標的タンパク質の探索
化合物SCを含む下記の構造を有するナフタレンスルホンアミド誘導体について、化合物Xとの相乗効果を調べた。
Test 11: Search for Target Protein of Compound SC The synergistic effect with compound X was investigated for naphthalenesulfonamide derivatives having the following structure, including compound SC.
化合物SCを含む下記の構造を有するナフタレンスルホンアミド誘導体について、化合物Xとの相乗効果を調べた。
pRL-CMVを恒常的に発現するHEK293A細胞を、いずれかのナフタレンスルホンアミド誘導体を図24に示す濃度で含む培地中、化合物X(100ng/ml)の存在下または非存在下で24時間培養し、ルシフェラーゼ活性を測定した。結果を図24に示す。CMVプロモーターの転写活性は、ナフタレンスルホンアミド誘導体のみでは変化しなかったが、ナフタレンスルホンアミド誘導体を化合物Xと併用すると増強された。ナフタレンスルホンアミド誘導体W-7はカルモジュリン阻害剤として利用されており、化合物SCおよびSC-9もカルモジュリン阻害剤として作用し、化合物Xとの相乗効果を示していると考えられる。
HEK293A cells constitutively expressing pRL-CMV were cultured for 24 hours in the presence or absence of compound X (100 ng/ml) in a medium containing any of the naphthalenesulfonamide derivatives at the concentrations shown in FIG. , luciferase activity was measured. The results are shown in FIG. The transcriptional activity of the CMV promoter was not changed by the naphthalenesulfonamide derivative alone, but was enhanced when the naphthalenesulfonamide derivative was used in combination with compound X. The naphthalenesulfonamide derivative W-7 has been utilized as a calmodulin inhibitor, and compounds SC and SC-9 also act as calmodulin inhibitors and are thought to exhibit a synergistic effect with compound X.
試験12:PKC活性化剤および遺伝子工学的PML発現によりCMVプロモーターの転写活性は増強される
pcDNAベクターのCMVプロモーターの下流に、RlucのcDNA、IRES、および、野生型PMLまたは欠損変異型PML(PMLΔ1~10またはPML-Ring)のcDNAを挿入し、発現コンストラクトを作製した(図25)。野生型PMLおよびPMLΔ1~10のコンストラクトをHEK293A細胞にトランスフェクションし、化合物Xの存在下または非存在下で24時間培養し、ルシフェラーゼ活性を測定した。結果を図26に示す。野生型PML、PMLΔ1およびPMLΔ6~10を導入された細胞では、対照(-)の細胞と比較して、化合物X存在下のルシフェラーゼ活性が高かった。この結果は、PMLが化合物Xにより増強されるCMVプロモーターの転写活性をさらに増強し、PMLの第1位~第46位を含むPro領域がその活性を担うことを示唆する。また、野生型PML、PMLΔ1およびPMLΔ6~10を導入された細胞では、化合物Xの非存在下でも、ルシフェラーゼ活性が対照(-)の細胞と比較して高かった。 Test 12: Transcriptional activity of CMV promoter is enhanced by PKC activator and genetically engineered PML expression. ~10 or PML-Ring) cDNA was inserted to generate an expression construct (Fig. 25). Wild-type PML and PMLΔ1-10 constructs were transfected into HEK293A cells, cultured in the presence or absence of compound X for 24 hours, and luciferase activity was measured. The results are shown in FIG. The luciferase activity in the presence of compound X was higher in cells transfected with wild-type PML, PMLΔ1 and PMLΔ6-10 compared to control (−) cells. This result suggests that PML further enhances the transcriptional activity of the CMV promoter enhanced by compound X, and that the Pro region containing positions 1-46 of PML is responsible for that activity. Also, in the cells into which wild-type PML, PMLΔ1 and PMLΔ6-10 were introduced, the luciferase activity was higher than in the control (−) cells even in the absence of compound X.
pcDNAベクターのCMVプロモーターの下流に、RlucのcDNA、IRES、および、野生型PMLまたは欠損変異型PML(PMLΔ1~10またはPML-Ring)のcDNAを挿入し、発現コンストラクトを作製した(図25)。野生型PMLおよびPMLΔ1~10のコンストラクトをHEK293A細胞にトランスフェクションし、化合物Xの存在下または非存在下で24時間培養し、ルシフェラーゼ活性を測定した。結果を図26に示す。野生型PML、PMLΔ1およびPMLΔ6~10を導入された細胞では、対照(-)の細胞と比較して、化合物X存在下のルシフェラーゼ活性が高かった。この結果は、PMLが化合物Xにより増強されるCMVプロモーターの転写活性をさらに増強し、PMLの第1位~第46位を含むPro領域がその活性を担うことを示唆する。また、野生型PML、PMLΔ1およびPMLΔ6~10を導入された細胞では、化合物Xの非存在下でも、ルシフェラーゼ活性が対照(-)の細胞と比較して高かった。 Test 12: Transcriptional activity of CMV promoter is enhanced by PKC activator and genetically engineered PML expression. ~10 or PML-Ring) cDNA was inserted to generate an expression construct (Fig. 25). Wild-type PML and PMLΔ1-10 constructs were transfected into HEK293A cells, cultured in the presence or absence of compound X for 24 hours, and luciferase activity was measured. The results are shown in FIG. The luciferase activity in the presence of compound X was higher in cells transfected with wild-type PML, PMLΔ1 and PMLΔ6-10 compared to control (−) cells. This result suggests that PML further enhances the transcriptional activity of the CMV promoter enhanced by compound X, and that the Pro region containing positions 1-46 of PML is responsible for that activity. Also, in the cells into which wild-type PML, PMLΔ1 and PMLΔ6-10 were introduced, the luciferase activity was higher than in the control (−) cells even in the absence of compound X.
また、PMLΔ9またはPML-Ringを含むコンストラクトをHEK293A細胞にトランスフェクションし、化合物X、SB、化合物SCまたはこれらの組合せの存在下または非存在下で24時間培養し、ルシフェラーゼ活性を測定した。結果を図27に示す。PMLΔ9またはPML-Ringを導入された細胞では、対照(Mock)の細胞と比較して、いずれの条件でもルシフェラーゼ活性が高かった。この結果は、PMLの第47位~第106位を含むRING領域も、化合物Xにより増強されるCMVプロモーターの転写活性をさらに増強することを示唆する。また、PMLΔ9またはPML-Ringを導入された細胞では、化合物Xの非存在下でも、ルシフェラーゼ活性が対照(-)の細胞と比較して高かった。
HEK293A cells were also transfected with constructs containing PMLΔ9 or PML-Ring, cultured for 24 hours in the presence or absence of compound X, SB, compound SC, or a combination thereof, and luciferase activity was measured. The results are shown in FIG. The cells into which PMLΔ9 or PML-Ring was introduced had higher luciferase activity under all conditions than the control (Mock) cells. This result suggests that the RING region encompassing PML positions 47-106 also enhances compound X-enhanced transcriptional activity of the CMV promoter. In addition, in the cells introduced with PMLΔ9 or PML-Ring, the luciferase activity was higher than the control (−) cells even in the absence of compound X.
試験13:PKC活性化剤およびRING領域を含むペプチドの遺伝子工学的発現によりCMVプロモーターの転写活性は増強される
RING領域は、各種のタンパク質間で共通の配列を有する。PMLのRING領域のアミノ酸配列をBLAST解析し、類似の配列を有するタンパク質として図28に示すタンパク質を同定した。これらのタンパク質のRING領域において、いくつかのシステイン残基およびヒスチジン残基に高い保存性が見られた。PMLのRING領域との類似性の程度が異なるタンパク質をいくつか選択し、以下の解析に用いた。 Test 13: Genetic Engineering Expression of a PKC Activator and a Peptide Containing the RING Region Enhances the Transcriptional Activity of the CMV Promoter The RING region has a common sequence among various proteins. BLAST analysis was performed on the amino acid sequence of the RING region of PML, and proteins shown in FIG. 28 were identified as proteins having similar sequences. Some cysteine and histidine residues were highly conserved in the RING regions of these proteins. Several proteins with different degrees of similarity to the PML RING region were selected and used for the following analysis.
RING領域は、各種のタンパク質間で共通の配列を有する。PMLのRING領域のアミノ酸配列をBLAST解析し、類似の配列を有するタンパク質として図28に示すタンパク質を同定した。これらのタンパク質のRING領域において、いくつかのシステイン残基およびヒスチジン残基に高い保存性が見られた。PMLのRING領域との類似性の程度が異なるタンパク質をいくつか選択し、以下の解析に用いた。 Test 13: Genetic Engineering Expression of a PKC Activator and a Peptide Containing the RING Region Enhances the Transcriptional Activity of the CMV Promoter The RING region has a common sequence among various proteins. BLAST analysis was performed on the amino acid sequence of the RING region of PML, and proteins shown in FIG. 28 were identified as proteins having similar sequences. Some cysteine and histidine residues were highly conserved in the RING regions of these proteins. Several proteins with different degrees of similarity to the PML RING region were selected and used for the following analysis.
pcDNAベクターのCMVプロモーターの下流に、RlucのcDNA、IRES、および、PML、TRIM3、TRIM56、COPI、Pex10、BRCA1またはHTLFのRING領域のcDNAを挿入し、発現コンストラクトを作製した(図29)。これらの発現コンストラクトをHEK293A細胞にトランスフェクションし、化合物Xの存在下または非存在下で24時間培養し、ルシフェラーゼ活性を測定した。結果を図30に示す。RING領域を導入された細胞では、対照(-)の細胞と比較して、化合物X存在下のルシフェラーゼ活性が高かった。この結果は、各種タンパク質のRING領域が、化合物Xにより増強されるCMVプロモーターの転写活性をさらに増強することを示唆する。また、RING領域を導入された細胞では、化合物Xの非存在下でも、対照(-)の細胞と比較してルシフェラーゼ活性が高かった。
The cDNA of Rluc, IRES, and the cDNA of the RING region of PML, TRIM3, TRIM56, COPI, Pex10, BRCA1 or HTLF were inserted downstream of the CMV promoter of the pcDNA vector to prepare an expression construct (Fig. 29). These expression constructs were transfected into HEK293A cells, cultured in the presence or absence of compound X for 24 hours, and luciferase activity was measured. The results are shown in FIG. The luciferase activity in the presence of compound X was higher in the cells introduced with the RING region than in the control (-) cells. This result suggests that the RING region of various proteins further enhances the CMV promoter transcriptional activity enhanced by Compound X. Also, in the cells into which the RING region was introduced, the luciferase activity was higher than in the control (-) cells even in the absence of compound X.
試験14:PMLによるCMVプロモーターの転写活性増強はmRNAの発現に依存する
pcDNAベクターのCMVプロモーターの下流に、RlucのcDNA、IRES、および、野生型PMLまたは欠損変異型PML(PMLΔ9、第2位に停止コドンが導入されたPMLΔ9、または、第7位に停止コドンが導入されたPMLΔ9)のcDNAを挿入し、発現コンストラクトを作製した(図31左)。また、pcDNAベクターのCMVプロモーターの下流に、RlucのcDNA、および、野生型PMLまたは欠損変異型PML(PMLΔ9、PMLΔ10またはPML-RING)のcDNAを挿入し、発現コンストラクトを作製した(図31右)。これらのコンストラクトを細胞に導入すると、PMLのmRNAは転写されるが、タンパク質には翻訳されない。これらのコンストラクトをHEK293A細胞にトランスフェクションし、化合物Xの存在下または非存在下で24時間培養し、ルシフェラーゼ活性を測定した。結果を図32に示す。いずれのコンストラクトでも、化合物Xの存在下および非存在下で、対照よりも高いルシフェラーゼ活性が見られた。この結果は、PMLによるCMVプロモーターの転写活性増強は、PMLのタンパク質ではなく、PMLのPro領域またはRING領域をコードするmRNAの発現に依存することを示唆する。 Test 14: Enhancement of transcriptional activity of CMV promoter by PML depends on mRNA expression. A cDNA of PMLΔ9 introduced with a stop codon or PMLΔ9 introduced with a stop codon at position 7) was inserted to prepare an expression construct (FIG. 31, left). In addition, the cDNA of Rluc and the cDNA of wild-type PML or defective mutant PML (PMLΔ9, PMLΔ10 or PML-RING) were inserted downstream of the CMV promoter of the pcDNA vector to prepare an expression construct (FIG. 31, right). . When these constructs are introduced into cells, the PML mRNA is transcribed but not translated into protein. These constructs were transfected into HEK293A cells, cultured in the presence or absence of Compound X for 24 hours, and luciferase activity was measured. The results are shown in FIG. Both constructs showed higher luciferase activity than the control in the presence and absence of compound X. This result suggests that enhancement of the transcriptional activity of the CMV promoter by PML depends on the expression of the mRNA encoding the PML Pro region or RING region, rather than the PML protein.
pcDNAベクターのCMVプロモーターの下流に、RlucのcDNA、IRES、および、野生型PMLまたは欠損変異型PML(PMLΔ9、第2位に停止コドンが導入されたPMLΔ9、または、第7位に停止コドンが導入されたPMLΔ9)のcDNAを挿入し、発現コンストラクトを作製した(図31左)。また、pcDNAベクターのCMVプロモーターの下流に、RlucのcDNA、および、野生型PMLまたは欠損変異型PML(PMLΔ9、PMLΔ10またはPML-RING)のcDNAを挿入し、発現コンストラクトを作製した(図31右)。これらのコンストラクトを細胞に導入すると、PMLのmRNAは転写されるが、タンパク質には翻訳されない。これらのコンストラクトをHEK293A細胞にトランスフェクションし、化合物Xの存在下または非存在下で24時間培養し、ルシフェラーゼ活性を測定した。結果を図32に示す。いずれのコンストラクトでも、化合物Xの存在下および非存在下で、対照よりも高いルシフェラーゼ活性が見られた。この結果は、PMLによるCMVプロモーターの転写活性増強は、PMLのタンパク質ではなく、PMLのPro領域またはRING領域をコードするmRNAの発現に依存することを示唆する。 Test 14: Enhancement of transcriptional activity of CMV promoter by PML depends on mRNA expression. A cDNA of PMLΔ9 introduced with a stop codon or PMLΔ9 introduced with a stop codon at position 7) was inserted to prepare an expression construct (FIG. 31, left). In addition, the cDNA of Rluc and the cDNA of wild-type PML or defective mutant PML (PMLΔ9, PMLΔ10 or PML-RING) were inserted downstream of the CMV promoter of the pcDNA vector to prepare an expression construct (FIG. 31, right). . When these constructs are introduced into cells, the PML mRNA is transcribed but not translated into protein. These constructs were transfected into HEK293A cells, cultured in the presence or absence of Compound X for 24 hours, and luciferase activity was measured. The results are shown in FIG. Both constructs showed higher luciferase activity than the control in the presence and absence of compound X. This result suggests that enhancement of the transcriptional activity of the CMV promoter by PML depends on the expression of the mRNA encoding the PML Pro region or RING region, rather than the PML protein.
本開示は、目的のタンパク質の生産量を高めることを可能にするため、バイオ医薬品の製造に利用できる。
The present disclosure can be used for the production of biopharmaceuticals, as it enables increased production of the protein of interest.
Claims (23)
- 目的のタンパク質の製造方法であって、SP1、CEBP、AP1、NF-κBおよびYY1から選択される少なくとも1つの転写因子の結合部位を含むプロモーターに作動可能に連結した目的のタンパク質をコードする核酸を含む細胞を、プロテインキナーゼC(PKC)を活性化する条件下で培養することを含み、
PKCを活性化する条件下での培養が、以下のいずれかである、方法:
(1)PKC活性化剤およびカルモジュリン阻害剤の存在下で前記細胞を培養する、
(2)活性化型PKCを前記細胞に発現させ、カルモジュリン阻害剤の存在下で培養する、
(3)Pro領域またはRING領域を含むペプチドをコードする核酸を前記細胞に発現させ、PKC活性化剤の存在下で培養する、
および、
(4)
から選択される化合物またはそのエステル、塩もしくは溶媒和物の存在下で前記細胞を培養する。 A method for producing a protein of interest, comprising providing a nucleic acid encoding the protein of interest operably linked to a promoter comprising a binding site for at least one transcription factor selected from SP1, CEBP, AP1, NF-κB and YY1. culturing the cell comprising, under conditions that activate protein kinase C (PKC),
A method, wherein culturing under conditions that activate PKC is any of the following:
(1) culturing said cells in the presence of a PKC activator and a calmodulin inhibitor;
(2) expressing activated PKC in the cells and culturing in the presence of a calmodulin inhibitor;
(3) expressing a nucleic acid encoding a peptide containing a Pro region or a RING region in said cells and culturing in the presence of a PKC activator;
and,
(4)
The cells are cultured in the presence of a compound selected from or an ester, salt or solvate thereof. - PKCを活性化する条件下での培養が、(1)PKC活性化剤およびカルモジュリン阻害剤の存在下で前記細胞を培養することである、請求項1に記載の方法。 The method according to claim 1, wherein the culturing under conditions that activate PKC is (1) culturing the cells in the presence of a PKC activator and a calmodulin inhibitor.
- PKCを活性化する条件下での培養が、(2)活性化型PKCを前記細胞に発現させ、カルモジュリン阻害剤の存在下で培養することである、請求項1に記載の方法。 The method according to claim 1, wherein the culturing under conditions that activate PKC is (2) expressing activated PKC in the cells and culturing in the presence of a calmodulin inhibitor.
- 前記細胞が、作動可能に連結されている、プロモーター、目的のタンパク質をコードする核酸、内部リボソーム進入部位または2Aペプチド配列、および、活性化型PKCをコードする核酸を含む発現コンストラクトを含む、請求項3に記載の方法。 4. The cell comprises an expression construct comprising, operably linked, a promoter, a nucleic acid encoding a protein of interest, an internal ribosome entry site or 2A peptide sequence, and a nucleic acid encoding an activated PKC. 3. The method described in 3.
- PKCを活性化する条件下での培養が、(3)Pro領域またはRING領域を含むペプチドをコードする核酸を細胞に発現させ、PKC活性化剤の存在下で培養することである、請求項1に記載の方法。 Claim 1, wherein the culturing under conditions that activate PKC is (3) allowing cells to express a nucleic acid encoding a peptide containing a Pro region or a RING region and culturing in the presence of a PKC activator. The method described in .
- RING領域を含むペプチドをコードする核酸を細胞に発現させる、請求項5に記載の方法。 The method according to claim 5, wherein a nucleic acid encoding a peptide containing a RING region is expressed in cells.
- RING領域が、TRIM13、LONRF3、TRIM47、RNF135、TRIM10、TRIM72、TRIM60、TRIM39、TRIM4、TRIM43B、TRIM43、TRIM25、TRIM26、TRIM31、HTLF、BRCA1、TRIM50、TRIM21、SSA1、TRIM5d、TRIM22、KIAA0182、TRIM65、RAG1、BFAR、Pex10、RNF8、RING2、COPI、TRIM2、TRIM3、SH3RF2、PMLまたはTRIM56のRING領域である、請求項6に記載の方法。 The RING region is TRIM13, LONRF3, TRIM47, RNF135, TRIM10, TRIM72, TRIM60, TRIM39, TRIM4, TRIM43B, TRIM43, TRIM25, TRIM26, TRIM31, HTLF, BRCA1, TRIM50, TRIM21, SSA1, TRIM5d, TRIM22, KIAA0182, TRIM65, 7. The method of claim 6, which is the RING region of RAG1, BFAR, Pex10, RNF8, RING2, COPI, TRIM2, TRIM3, SH3RF2, PML or TRIM56.
- RING領域が、PML、TRIM3、TRIM56、COPI、Pex10、BRCA1またはHTLFのRING領域である、請求項6または7に記載の方法。 8. The method according to claim 6 or 7, wherein the RING region is the RING region of PML, TRIM3, TRIM56, COPI, Pex10, BRCA1 or HTLF.
- Pro領域を含むペプチドをコードする核酸を細胞に発現させる、請求項5に記載の方法。 The method according to claim 5, wherein a nucleic acid encoding a peptide containing a Pro region is expressed in cells.
- 前記細胞が、作動可能に連結されている、プロモーター、目的のタンパク質をコードする核酸、および、Pro領域またはRING領域を含むペプチドをコードする核酸を含む発現コンストラクトを含む、請求項5~9のいずれかに記載の方法。 10. Any of claims 5-9, wherein the cell comprises an expression construct comprising, operatively linked, a promoter, a nucleic acid encoding a protein of interest, and a nucleic acid encoding a peptide comprising a Pro region or a RING region. The method described in Crab.
- PKC活性化剤が、式(I):
R1は、H、ハロゲン、-OH、C1-6アルキル、C2-6アルケニル、C2-6アルキニル、C1-6アルコキシ、C6-14アリールまたは-OC(O)R3であり、ここで、C1-6アルキル、C2-6アルケニル、C2-6アルキニル、C1-6アルコキシまたはアリールは、同一または異なる、1個~3個のハロゲンにより置換されていてもよく、
R2は、C6-12アルキル、C6-12アルケニル、C6-12アルキニルまたはC6-12アルコキシであり、ここで、C6-12アルキル、C6-12アルケニル、C6-12アルキニルまたはC6-12アルコキシは、同一または異なる、1個~3個のハロゲンにより置換されていてもよく、
R3は、C1-6アルキル、C2-6アルケニル、C2-6アルキニル、アミノまたはC6-14アリールであり、ここでC1-6アルキル、C2-6アルケニル、C2-6アルキニルまたはC6-14アリールは、同一または異なる、1個~3個のハロゲンにより置換されていてもよい}、
式(II):
R4は、H、ハロゲン、-OH、C1-18アルキル、C2-18アルケニル、C2-18アルキニル、C1-18アルコキシまたは-OC(O)R6であり、ここで、C1-18アルキル、C2-18アルケニル、C2-18アルキニルまたはC1-18アルコキシは、同一または異なる、1個~3個のハロゲンにより置換されていてもよく、
R5は、C1-6アルキル、C2-6アルケニル、C2-6アルキニルまたはアミノであり、ここで、C1-6アルキル、C2-6アルケニルまたはC2-6アルキニルは、同一または異なる、1個~3個のハロゲンにより置換されていてもよく、
R6は、C1-18アルキル、C2-18アルケニル、C2-18アルキニルまたはアミノであり、ここでC1-18アルキル、C2-18アルケニルまたはC2-18アルキニルは、同一または異なる、1個~3個のハロゲンにより置換されていてもよい}、
または、
式(III):
R7は、H、C1-18アルキル、C2-18アルケニル、C2-18アルキニルまたは-C(O)R9であり、ここで、C1-18アルキル、C2-18アルケニルまたはC2-18アルキニルは、同一または異なる、1個~3個のハロゲンにより置換されていてもよく、
R8は、H、C1-18アルキル、C2-18アルケニル、C2-18アルキニルまたは-C(O)R9であり、ここで、C1-18アルキル、C2-18アルケニルまたはC2-18アルキニルは、同一または異なる、1個~3個のハロゲンにより置換されていてもよく、
R9は、C1-18アルキル、C2-18アルケニル、C2-18アルキニルまたはアミノであり、ここでC1-18アルキル、C2-18アルケニルまたはC2-18アルキニルは、同一または異なる、1個~3個のハロゲンにより置換されていてもよい}
の化合物またはそのエステル、塩もしくは溶媒和物である、請求項2および5~10のいずれかに記載の方法。 The PKC activator has formula (I):
R 1 is H, halogen, —OH, C 1-6 alkyl, C 2-6 alkenyl, C 2-6 alkynyl, C 1-6 alkoxy, C 6-14 aryl or —OC(O)R 3 , wherein C 1-6 alkyl, C 2-6 alkenyl, C 2-6 alkynyl, C 1-6 alkoxy or aryl are optionally substituted by 1 to 3 halogens, the same or different,
R 2 is C 6-12 alkyl, C 6-12 alkenyl, C 6-12 alkynyl or C 6-12 alkoxy, where C 6-12 alkyl, C 6-12 alkenyl, C 6-12 alkynyl or C 6-12 alkoxy is optionally substituted by 1 to 3 halogens, which may be the same or different;
R 3 is C 1-6 alkyl, C 2-6 alkenyl, C 2-6 alkynyl, amino or C 6-14 aryl where C 1-6 alkyl, C 2-6 alkenyl, C 2-6 alkynyl or C 6-14 aryl optionally substituted by 1 to 3 halogens, the same or different},
Formula (II):
R 4 is H, halogen, —OH, C 1-18 alkyl, C 2-18 alkenyl, C 2-18 alkynyl, C 1-18 alkoxy or —OC(O)R 6 where C 1 -18 alkyl, C 2-18 alkenyl, C 2-18 alkynyl or C 1-18 alkoxy is optionally substituted by 1 to 3 halogens, which may be the same or different;
R 5 is C 1-6 alkyl, C 2-6 alkenyl, C 2-6 alkynyl or amino, wherein C 1-6 alkyl, C 2-6 alkenyl or C 2-6 alkynyl are the same or optionally substituted by 1 to 3 different halogens,
R 6 is C 1-18 alkyl, C 2-18 alkenyl, C 2-18 alkynyl or amino, wherein the C 1-18 alkyl, C 2-18 alkenyl or C 2-18 alkynyl are the same or different , optionally substituted by 1 to 3 halogens},
or,
Formula (III):
R 7 is H, C 1-18 alkyl, C 2-18 alkenyl, C 2-18 alkynyl or —C(O)R 9 where C 1-18 alkyl, C 2-18 alkenyl or C 2-18 alkynyl is optionally substituted by 1 to 3 halogens, which may be the same or different;
R 8 is H, C 1-18 alkyl, C 2-18 alkenyl, C 2-18 alkynyl or —C(O)R 9 where C 1-18 alkyl, C 2-18 alkenyl or C 2-18 alkynyl is optionally substituted by 1 to 3 halogens, which may be the same or different;
R 9 is C 1-18 alkyl, C 2-18 alkenyl, C 2-18 alkynyl or amino, wherein the C 1-18 alkyl, C 2-18 alkenyl or C 2-18 alkynyl are the same or different , optionally substituted by 1 to 3 halogens}
or an ester, salt or solvate thereof. - PKC活性化剤が、化合物X、化合物#1~#7、TPA、プロストラチン、(-)-インドラクタムV、ホルボール12,13-ジブチラート、インゲノール3-アンゲラートおよび(2S,5S)-(E,E)-8-(5-(4-(トリフルオロメチル)フェニル)-2,4-ペンタジエノイルアミノ)ベンゾラクタムから選択される化合物またはそのエステル、塩もしくは溶媒和物である、請求項2および5~10のいずれかに記載の方法。 PKC activators are compound X, compounds #1-#7, TPA, prostratin, (−)-indolactam V, phorbol 12,13-dibutyrate, ingenol 3-angelate and (2S,5S)-(E, E) is a compound selected from -8-(5-(4-(trifluoromethyl)phenyl)-2,4-pentadienoylamino)benzolactam or its ester, salt or solvate, claim 2 and the method according to any one of 5-10.
- PKC活性化剤が、化合物Xおよび化合物#1~#7から選択される化合物またはそのエステル、塩もしくは溶媒和物である、請求項2および5~10のいずれかに記載の方法。 The method according to any one of claims 2 and 5 to 10, wherein the PKC activator is a compound selected from compound X and compounds #1 to #7 or an ester, salt or solvate thereof.
- カルモジュリン阻害剤が、
式(IV)
nは1~8の整数であり、
Rは、C1-6アルキル、C2-6アルケニル、C2-6アルキニル、C1-6アルコキシ、C6-14アリール、アミノ、ヒドロキシ、COOHまたはCOOR’であり、ここでR’はC1-6アルキルである}
の化合物またはそのエステル、塩もしくは溶媒和物である、請求項2~4のいずれかに記載の方法。 A calmodulin inhibitor
Formula (IV)
n is an integer from 1 to 8,
R is C 1-6 alkyl, C 2-6 alkenyl, C 2-6 alkynyl, C 1-6 alkoxy, C 6-14 aryl, amino, hydroxy, COOH or COOR', where R' is C 1-6 alkyl}
The method according to any one of claims 2 to 4, which is a compound of or an ester, salt or solvate thereof. - PKCを活性化する条件下での培養が、(4)化合物Xおよび化合物#1~#7から選択される化合物またはそのエステル、塩もしくは溶媒和物の存在下で前記細胞を培養することである、請求項1に記載の方法。 The culturing under conditions that activate PKC is (4) culturing the cells in the presence of a compound selected from compound X and compounds #1 to #7 or esters, salts or solvates thereof. A method according to claim 1.
- ヒストンデアセチラーゼ阻害剤の存在下で細胞を培養する、請求項1~15のいずれかに記載の方法。 The method according to any one of claims 1 to 15, wherein the cells are cultured in the presence of a histone deacetylase inhibitor.
- ヒストンデアセチラーゼ阻害剤が酪酸ナトリウムである、請求項16に記載の方法。 The method of claim 16, wherein the histone deacetylase inhibitor is sodium butyrate.
- 目的の遺伝子の転写を増強する方法であって、SP1、CEBP、AP1、NF-κBおよびYY1から選択される少なくとも1つの転写因子の結合部位を含むプロモーターに作動可能に連結した当該遺伝子の核酸を含む細胞を、PKCを活性化する条件下で培養することを含み、
PKCを活性化する条件下での培養が、以下のいずれかである、方法:
(1)PKC活性化剤およびカルモジュリン阻害剤の存在下で前記細胞を培養する、
(2)活性化型PKCを前記細胞に発現させ、カルモジュリン阻害剤の存在下で培養する、
(3)Pro領域またはRING領域を含むペプチドをコードする核酸を前記細胞に発現させ、PKC活性化剤の存在下で培養する、および、
(4)
から選択される化合物またはそのエステル、塩もしくは溶媒和物の存在下で前記細胞を培養する。 A method of enhancing transcription of a gene of interest comprising providing nucleic acid of the gene operably linked to a promoter comprising a binding site for at least one transcription factor selected from SP1, CEBP, AP1, NF-κB and YY1. culturing the cell comprising, under conditions that activate PKC;
A method, wherein culturing under conditions that activate PKC is any of the following:
(1) culturing said cells in the presence of a PKC activator and a calmodulin inhibitor;
(2) expressing activated PKC in the cells and culturing in the presence of a calmodulin inhibitor;
(3) expressing a nucleic acid encoding a peptide containing a Pro region or a RING region in said cells and culturing in the presence of a PKC activator;
(4)
The cells are cultured in the presence of a compound selected from or an ester, salt or solvate thereof. - 目的のタンパク質を製造するためのキットであって、
(1)PKC活性化剤およびカルモジュリン阻害剤、
(2)活性化型PKCをコードする核酸およびカルモジュリン阻害剤、
(3)PKC活性化剤およびPro領域またはRING領域を含むペプチドをコードする核酸、または、
(4)
から選択される化合物またはそのエステル、塩もしくは溶媒和物、
を含み、
目的のタンパク質の製造が、SP1、CEBP、AP1、NF-κBおよびYY1から選択される少なくとも1つの転写因子の結合部位を含むプロモーターに作動可能に連結した目的のタンパク質をコードする核酸を含む細胞を培養することを含む、キット。 A kit for producing a protein of interest, comprising:
(1) PKC activators and calmodulin inhibitors,
(2) a nucleic acid encoding activated PKC and a calmodulin inhibitor;
(3) a nucleic acid encoding a peptide comprising a PKC activator and a Pro region or a RING region, or
(4)
a compound or an ester, salt or solvate thereof selected from
including
Production of the protein of interest comprises a cell comprising a nucleic acid encoding the protein of interest operably linked to a promoter comprising a binding site for at least one transcription factor selected from SP1, CEBP, AP1, NF-κB and YY1. A kit comprising culturing. - 作動可能に連結されている、SP1、CEBP、AP1、NF-κBおよびYY1から選択される少なくとも1つの転写因子の結合部位を含むプロモーター、タンパク質をコードする核酸、内部リボソーム進入部位または2Aペプチド配列、および、活性化型PKCをコードする核酸を含む、発現コンストラクト。 a promoter comprising a binding site for at least one transcription factor selected from SP1, CEBP, AP1, NF-κB and YY1, operably linked, a nucleic acid encoding a protein, an internal ribosome entry site or a 2A peptide sequence; and an expression construct comprising a nucleic acid encoding activated PKC.
- 作動可能に連結されている、SP1、CEBP、AP1、NF-κBおよびYY1から選択される少なくとも1つの転写因子の結合部位を含むプロモーター、タンパク質をコードする核酸、および、Pro領域またはRING領域を含むペプチドをコードする核酸を含む、発現コンストラクト。 a promoter comprising a binding site for at least one transcription factor selected from SP1, CEBP, AP1, NF-κB and YY1, operably linked, a nucleic acid encoding a protein, and a Pro region or a RING region An expression construct comprising a nucleic acid encoding a peptide.
- 目的のタンパク質の製造方法であって、作動可能に連結されている、SP1、CEBP、AP1、NF-κBおよびYY1から選択される少なくとも1つの転写因子の結合部位を含むプロモーター、目的のタンパク質をコードする核酸、および、Pro領域またはRING領域を含むペプチドをコードする核酸を含む発現コンストラクトを細胞に発現させ、培養することを含む、方法。 A method for producing a protein of interest, comprising: a promoter comprising a binding site for at least one transcription factor selected from SP1, CEBP, AP1, NF-κB and YY1, operably linked, encoding the protein of interest and expressing in a cell and culturing an expression construct comprising a nucleic acid encoding a peptide comprising a Pro region or a RING region.
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